Biomedical subjects

K P Ponder

Publications and source records attributed to K P Ponder.

At least 19 recordsLinked to original sources

PubMed2001

Lipopolysaccharide potentiates the effect of hepatocyte growth factor upon replication in lung, thyroid, spleen, and colon in rats in vivo.

Induction of replication may potentiate in vivo gene therapy, as some viral vectors only transduce dividing cells. Hepatocyte growth factor (HGF) increases the percentage of replicating hepatocytes to 18-fold that in normal rats, and lipopolysaccharide (LPS) modestly potentiates this effect. In this study, the effect of iv HGF upon replication in other organs was determined. HGF at 10 mg/kg resulted in replication that was < or =3-fold that of normal rats in alveolar and proximal renal tubular cells. HGF alone had no effect upon replication of epithelial cells from the bronchi, thyroid, pancreas, or colon or upon cells from the muscle, pancreatic islets, spleen, blood vessels, or thymus. HGF and LPS at 5 mg/kg resulted in replication that was 9-fold that of normal rats in alveolar cells, 25-fold in bronchial epithelial cells, 4-fold in thyroid epithelial cells, 1.5-fold in the red pulp of the spleen, and 2-fold in colonic epithelial cells. The synergistic effect may be due to the fact that LPS upregulated the HGF receptor c-met in thyroid, spleen, and colon. We conclude that iv administration of HGF alone is relatively specific for inducing hepatocyte replication and would allow selective gene transfer into the liver.

Animals↗

PubMed2001

Lipopolysaccharide results in a marked decrease in hepatocyte nuclear factor 4 alpha in rat liver.

The acute-phase response can result in decreased liver-specific functions and death as a result of liver failure. We show here that lipopolysaccharide (LPS), an endotoxin that induces the acute-phase response, results in a marked decrease in the major isoforms of the transcription factor, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), in livers of rats. HNF-4 alpha is a nuclear receptor that is critical for the expression of several liver-specific genes. This decrease in HNF-4 alpha is primarily the result of a posttranscriptional mechanism, because mRNA levels are normal, and there are no major changes in the splicing patterns. This decrease was of functional significance, because expression of a gene that is highly dependent on HNF-4 alpha, HNF-1 alpha, was reduced. Interleukin-1 beta (IL-1 beta) is a cytokine whose levels are increased in vivo in response to LPS. IL-1 beta resulted in a decrease in HNF-4 alpha levels in HepG2 cells. This IL-1 beta-induced decrease was likely caused by degradation via the proteasome, because it was prevented by the addition of the proteasome inhibitor, MG132. We conclude that the decrease in HNF-4 alpha that occurs in vivo after the administration of LPS may be the result of IL-1 beta-induced degradation, and likely contributes to the liver insufficiency that occurs. IL-1 beta antagonists or proteasome inhibitors might increase HNF-4 alpha protein levels in the acute-phase response, which could result in increased liver function and survival.

Animals↗

PubMed2000

Delivery of a retroviral vector expressing human beta-glucuronidase to the liver and spleen decreases lysosomal storage in mucopolysaccharidosis VII mice.

Mucopolysaccharidosis VII (MPS VII) is caused by beta-glucuronidase (beta-gluc) deficiency and results in lysosomal storage due to the inability to degrade glycosaminoglycans. Transfer of a beta-gluc gene into the liver reduces hepatic pathology as well as storage in other organs via uptake of secreted protein. A Moloney murine leukemia-based retroviral vector expressing the human beta-gluc cDNA was injected intravascularly into MPS VII mice during hepatocyte replication, which was induced with im injection of an adenoviral vector that transiently expressed hepatocyte growth factor (Ad.CMV. HGF). This procedure resulted in transduction of approximately 1% of hepatocytes, 1% of normal liver enzyme activity, and a reduction in lysosomal storage in the liver at 3.5 months. Surprisingly, controls that received retroviral vector without HGF had transduction of nonparenchymal cells in the liver, significant levels of enzyme and RNA in the liver at 2 but not 3.5 months, and reduced lysosomal storage at 3.5 months. Transduction was also achieved in the replicating cells of the spleen, where lysosomal storage was reduced. An approach using a retroviral vector without a growth factor might temporarily reduce lysosomal storage in the liver and spleen in humans. Addition of HGF might be used to augment and prolong gene transfer.

Animals↗

PubMed2000

Lovastatin decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats.

BACKGROUND/AIMS: Liver insufficiency occurs when the liver cannot perform critical functions such as ammonia metabolism, gluconeogenesis, or production of coagulation factors The hypothesis of this study was that decreased function of existing hepatocytes may contribute to hepatic failure, and that the function of these cells might be increased pharmacologically. Lovastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor that inhibits cholesterol biosynthesis and affects the activity of some signal transduction pathways and liver transcription factors. Changes in hepatic transcription factors during liver regeneration might result in decreased liver functions, and lovastatin might prevent these changes METHODS: Rats received 90% partial hepatectomy (90% PH), and either lovastatin or vehicle alone daily. Survival and liver functions were assessed. RESULTS: Lovastatin increased survival to 58% (vs. 6% in controls that received 90% PH without drug), decreased the peak ammonia level to 427 microM (vs. 846 microM in controls), increased the nadir of glucose to 88 mg/dl (vs. 57 mg/dl in controls), decreased the peak prothrombin time to 23 s (vs 29 s in controls), and decreased the peak activated partial thromboplastin time to 29 s (vs. 39 s in controls). The full survival and metabolic benefits were observed when lovastatin was started at 30 min after 90% PH, but lovastatin was less efficacious when started at later times. CONCLUSIONS: Lovastatin increases the function of existing hepatocytes and might be used to improve liver function after extensive hepatic resection.

Ammonia↗

PubMed1999

Intramuscular injection of an adenoviral vector expressing hepatocyte growth factor facilitates hepatic transduction with a retroviral vector in mice.

Retroviral vectors can result in therapeutic and stable levels of expression of proteins from the liver. However, most retroviral vectors transduce only dividing cells, and hepatocytes are normally quiescent. The goal of this study was to determine if an adenoviral vector could transiently express hepatocyte growth factor (HGF) in order to induce hepatocyte replication and facilitate retroviral vector transduction of the liver. Intramuscular injection of an adenoviral vector that expressed human HGF from the cytomegalovirus promoter (Ad.CMV.HGF) resulted in moderate levels of HGF in blood and liver, and replication of 3 to 12% of hepatocytes. No cytopathic effect was observed in the liver, and a control adenoviral vector induced no or lower levels of replication. When a retroviral vector expressing beta-galactosidase cDNA was injected into a peripheral vein during the peak period of hepatocyte replication induced by intramuscularly administered Ad.CMV.HGF, 8% of hepatocytes were transduced. We conclude that intramuscular injection of Ad.CMV.HGF is a safe and effective way to induce transient systemic expression of HGF and hepatocyte replication, and to facilitate transduction of hepatocytes with a retroviral vector.

Adenoviridae↗

PubMed1999

Lipopolysaccharide potentiates the effect of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity.

The liver regenerates by replication of differentiated hepatocytes after damage or removal of part of the liver. Although several growth factors and signaling pathways are activated during regeneration, it is unclear as to which of these are essential for hepatocyte replication. We show here that low- (1 mg/kg) and high- (10 mg/kg) dose hepatocyte growth factor (HGF) induced replication of 2.1% and 11.1% of hepatocytes in rats, respectively. Lipopolysaccharide (LPS), an inducer of the acute phase response, augmented hepatocyte replication in response to low- and high-dose HGF by 4- and 2-fold, respectively. HGF alone induced moderate levels of c-Jun-N-terminal kinase (JNK) and p44/p42 mitogen-activated protein kinase (MAPK), resulting in moderate levels of AP-1-DNA binding activity. The combination of LPS + HGF increased JNK and AP-1-DNA binding activity more than levels seen with LPS or HGF alone. The activation of Stat3 that was observed after administration of LPS + HGF, but not HGF alone, could contribute to increased transcription of AP-1 components. Because phosphorylation of the c-Jun component of AP-1 by JNK increases its ability to activate transcription, the AP-1 in hepatocytes from animals treated with LPS + HGF may be more active than in rats treated with LPS or HGF alone. LPS may contribute to hepatocyte replication by potentiating the effect of HGF on the activation of both AP-1-DNA binding and transcriptional activity.

Acute-Phase Proteins↗

PubMed1999

Systemic gene therapy for cardiovascular disease.

Systemic gene therapy involves the transfer into the body of a gene whose protein product reaches the blood and has a beneficial effect on a patient. Both retroviral and adenovirus-associated viral vectors have resulted in stable but only moderate systemic levels of blood proteins. Adenoviral vectors have resulted in very high levels of expression that diminishes over days or weeks. Hepatic gene therapy has achieved levels of the anticoagulant protein C in blood that would protect against spontaneous thromboses in homozygous protein-C deficiency, and levels of tissue plasminogen activator that can lyse pulmonary emboli. Hypercholesterolemia has been ameliorated transiently by transfer of the low-density lipoprotein receptor gene into the livers of animals with familial hypercholesterolemia or by promoting lipid transfer via a variety of alternative mechanisms. Hypertension has been reduced by the transfer of genes for kallikrein or atrial natriuretic peptide into the liver, or by expressing antisense for the angiotensin II type I receptor after intravenous injection in neonates. Finally, fasting but not fed hyperglycemia has been ameliorated in animal models of diabetes by transfer of an insulin gene into the liver or by expression of insulin from implanted fibroblasts. Gene therapy has the potential to treat these cardiovascular diseases. However, improvements in levels of long-term expression and the ability to regulate expression in response to physiologic changes will be required before this approach will be implemented for most of these disorders in humans.

Animals↗

PubMed1999

Embolization of portal vein branches induces hepatocyte replication in swine: a potential step in hepatic gene therapy.

PURPOSE: To determine whether embolization of portal vein branches would stimulate hepatocyte replication in pigs. MATERIALS AND METHODS: The portal vein branches supplying 50%-70% of the liver were embolized in eight pigs by using a combination of coils and polyvinyl alcohol particles. The extent of embolization was assessed at portography in all animals and at computed tomography in one animal. Hepatocyte replication was determined by calculating the percentage of cells that incorporated bromodeoxyuridine into their nuclei. Animals survived up to 35 days after the procedure. RESULTS: Embolization of the portal vein branches supplying the left and median lobes caused transient increases of less than 70% in portal vein pressures and of less than 100% in liver enzyme levels. Indocyanine green clearance was measured in two animals and decreased less than 50%. The percentage of replicating hepatocytes in the nonembolized lobe was 0% on day 0, 7% on day 2, 14% on day 7, and 2% on day 12. CONCLUSION: Substantial hepatocyte replication occurred 2-7 days after embolization of portal vein branches. Further research will help determine if this procedure can facilitate retroviral transduction in large animals. If successful, the low morbidity of this method may allow its use in humans for gene therapy.

Animals↗

PubMed1998

Therapeutic levels of human protein C in rats after retroviral vector-mediated hepatic gene therapy.

Protein C deficiency results in a thrombotic disorder that might be treated by expressing a normal human protein C (hPC) gene in patients. An amphotropic retroviral vector with a liver-specific promoter and the hPC cDNA was delivered to rat hepatocytes in vivo during liver regeneration. Expression of hPC varied from 55 to 203 ng/ml (1.3-5.0% of normal) for 2 wk after transduction. Expression increased to an average of 900 ng/ml (22% of normal) in some rats and was maintained at stable levels for 1 yr. All of these rats developed anti-hPC antibodies and exhibited a prolonged hPC half-life in vivo. The hPC was functional as determined by a chromogenic substrate assay after immunoprecipitation. We conclude that most rats achieved hPC levels that would prevent purpura fulminans, and that hepatic gene therapy might become a viable treatment for patients with severe homozygous hPC deficiency. Anti-hPC antibodies increased the hPC half-life and plasma levels in some rats, but did not interfere with its functional activity. Thus, the development of antibodies against a plasma protein does not necessarily abrogate its biological effect in gene therapy experiments.

Animals↗

PubMed1997

Therapeutic levels of functional human factor X in rats after retroviral-mediated hepatic gene therapy.

Factor X deficiency results in a rare but serious bleeding disorder that might be treated by expressing a normal factor X gene in patients. We generated an amphotropic retroviral vector with the human FX cDNA and delivered it to rat hepatocytes in vivo during liver regeneration. The human alpha1-antitrypsin promoter was chosen to direct expression because it was the most efficient of several tested in yielding expression of alpha1-antitrypsin protein from a retroviral vector in hepatocytes in vivo. We achieved expression of factor X in four rats at levels sufficient to maintain hemostasis in humans (10% to 43% of normal). The factor X was determined to be functional by using a chromogenic substrate assay after immunoprecipitation with human specific antibodies. Expression of factor X remained stable for more than 10 months in two rats. It is likely that expression will be maintained for the life of the animals, because retroviral vectors integrate into the chromosome and hepatocytes are long-lived. The high and stable levels of expression achieved using this liver-specific promoter overcomes one of the two major obstacles to successful human gene therapy for hemophilia.

3T3 Cells↗

PubMed1996

Portal branch occlusion safely facilitates in vivo retroviral vector transduction of rat liver.

Hepatic gene therapy might correct the clinical manifestations of several genetic disorders in patients. Although retroviral vectors with a strong liver-specific promoter can result in stable and therapeutic levels of expression of genes from the liver, application of these techniques in humans is limited by the need to perform one or more invasive procedures to achieve ex vivo or in vivo transduction of hepatocytes. In vivo delivery involves injection of retrovirus into the portal vein during liver regeneration. Although transduction is efficient and specific for the liver, induction of hepatocyte replication requires a 70% partial hepatectomy or administration of a liver toxin. An alternative method for inducing hepatocyte replication is to occlude branches of the portal vein. This results in apoptosis of hepatocytes in the occluded lobes and compensatory replication of the hepatocytes in the nonoccluded lobes. We demonstrate here that portal branch occlusion is nearly as effective as partial hepatectomy at facilitating retroviral vector transduction in vivo and has a lower morbidity. Portal branch occlusion could be performed in larger animals by minimally invasive techniques and has been used safely to treat human patients with liver cancer. Portal branch occlusion might ultimately be used in humans to facilitate retroviral vector transduction in vivo for the treatment of genetic diseases.

Alanine Transaminase↗

PubMed1996

Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo.

Hepatic gene therapy could improve the treatment of many inherited disorders. Although retroviral vectors result in long-term expression in hepatocytes in vivo, their low level of expression currently precludes most clinical applications. Four copies of the liver-specific apolipoprotein E (ApoE) enhancer were placed upstream of the human alpha 1-antitrypsin (hAAT) promoter in either orientation into a retroviral vector with a complete long terminal repeat (LTR) and the hAAT cDNA to generate ApoE(+)hAAT-LTR and ApoE(-)hAAT-LTR. In addition, the ApoAI promoter was placed upstream of the hAAT cDNA in a similar retroviral vector backbone. Amphotropic retroviral vectors were transferred into regenerating rat liver cells in vivo by intraportal injection. ApoE(-)hAAT-LTR and ApoE(+)hAAT-LTR led to average hAAT levels of 5 micrograms/ml (0.5% of normal levels of a very abundant protein), and 2.5 micrograms/ml, respectively, which was stable for at least 10 months after transduction. This level of serum hAAT was > 25-fold higher than what was observed from the ApoAI promoter used in this study. Serum levels of hAAT were > 15-fold higher than what was observed from retroviral vectors containing the hAAT cDNA that were analyzed previously by this lab. In some cases, improved expression was due to the promoter chosen. In other cases, the increase in expression was primarily due to the higher titers obtained by using a retroviral backbone with an intact LTR as opposed to a vector with a deletion in the LTR. The increased expression levels observed from this enhancer/promoter combination in an intact retroviral backbone may enable one to achieve therapeutic levels of clinically important genes from a retroviral vector in liver cells of animals.

Animals↗

PubMed1996

Retroviral vector sequences may interact with some internal promoters and influence expression.

Although retroviral vectors show promise for gene therapy, their expression in animals has been low. An improved understanding of how promoters function from a retroviral vector should facilitate the design of improved vectors. In this study, liver-specific promoters were cloned into a retroviral vector and expression from the retroviral long terminal repeat (LTR) and the internal promoter was analyzed. In addition, oligomerized liver-specific transcription factor binding sites were placed upstream of each promoter in an attempt to increase expression further. Additional oligomerized binding sites only increased expression slightly or inhibited expression in hepatoma cells, suggesting that this is not an effective way to increase expression from a retroviral vector. Unexpectedly, the liver-specific albumin promoter was expressed at high levels from a retroviral vector in fibroblasts, suggesting that retroviral elements functioned as an enhancer. Furthermore, the addition of HNF-4 binding sites adjacent to the albumin promoter inhibited both the LTR and albumin promoter in fibroblasts, an effect that was probably mediated by inhibitory proteins present in nonhepatic cells that can bind to HNF-4 sites. These results suggest that both positive and negative influences can be transmitted between the LTR and the albumin promoter. In contrast, the liver-specific human alpha 1-antitrypsin promoter did not appear to interact with the LTR by either of these criteria. Retroviral vectors have sequences that may inhibit expression of the LTR and some internal promoters in vivo. We hypothesize that internal promoters that do not interact with the LTR in tissue culture will be resistant to inhibitory effects of retroviral sequences in vivo.

Albumins↗

PubMed1996

Alterations in enzymatic functions in hepatocytes and hepatocellular carcinomas from Ras-transduced livers resemble the effects of insulin.

An understanding of how oncogenes affect differentiated liver functions might lead to improved treatments for liver cancer or other disorders where liver-specific functions are compromised. A retroviral vector that coexpressed beta-galactosidase (beta-gal) and activated Ras genes (Ras-gal) was transduced into a small fraction of adult rat hepatocytes in vivo. Hepatocytes from Ras-gal-transduced diethylnitrosamine-untreated livers and hepatocellular carcinomas (HCC) from Ras-gal-transduced diethylnitrosamine-treated rats were analyzed for liver functions by performing histochemical assays on liver sections. Ras-gal-transduced hepatocytes failed to express gluconeogenic, ketogenic, and urea pathway enzymes. In contrast, several enzymes involved in fat synthesis were strongly activated, and microvesicular fat accumulated. These metabolic changes are induced in normal livers by insulin, a hormone that activates p21-ras. The deregulation of p21-ras may inhibit these liver-specific functions and may induce fat synthesis in both malignant and nonmalignant liver diseases. Furthermore, treatment with drugs that inhibit the attachment of p21-ras to the plasma membrane might reverse these changes. The alterations in enzymatic functions in the HCCs were similar to those observed in the hepatocytes, although each of the two cancers had a region that abruptly lost its expression of liver-specific enzymes and acquired the expression of genes that are more characteristic of oval or bile ductule cells. This suggests that a single genetic event in a malignant cell may dramatically alter its apparent phenotype. The identification of this putative gene might lead to insights into the regulation of the phenotype of normal cells in the liver.

3T3 Cells↗

PubMed1996

Analysis of liver development, regeneration, and carcinogenesis by genetic marking studies.

The mechanism of generating new hepatocytes and bile ductule cells in the liver has been controversial. Oval cells are found in the periportal region under some circumstances and may represent multipotent stem cells. The role of stem cells in generating new liver cells in normal and pathological conditions is unclear, however. Genetic marking can be used to determine the ability of a particular cell to replicate and to migrate. Cells of known lineage are marked at an initial time point, and their developmental potential determined by the cluster size, position, and phenotype of marked cells at a later time point. Recently, genetic marking studies have demonstrated that the hepatocyte itself is the source of new hepatocytes in the normal postnatal liver and that daughter cells do not migrate. These studies have also demonstrated that the hepatocyte can replicate extensively when stimulated. Finally, genetic marking studies suggest that either hepatocytes or oval cells can develop into a hepatocellular carcinoma or cholangiocarcinoma if a sufficient number of genetic mutations accumulate. The implications of these results for hepatic gene therapy, treatment of liver insufficiency states, and liver cancer are discussed. Future genetic marking studies may help to address some remaining questions in liver biology.

Animals↗

PubMed1995

Ras-transduced diethylnitrosamine-treated hepatocytes develop into cancers of mixed phenotype in vivo.

The cell of origin of hepatocellular carcinoma (HCC) is controversial. A method for marking cells of different lineages in vivo and then determining their carcinogenic potential should resolve this issue. A retroviral vector expressing activated ras and beta-gal genes (Ras-gal) was transferred into adult rat hepatocytes in vivo, and some animals were treated with diethylnitrosamine (DEN). Bile ductule cells and the putative stem cells of the liver (the oval cells) did not appear to be transduced by this method. At 1 month after transfer, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining was performed on transduced rat livers to determine the blue cluster size. Eight % of the clusters in Ras-gal-transduced, DEN-treated livers contained at least twice as many cells as the largest cluster in Ras-gal-transduced, DEN-untreated rats, demonstrating that they had acquired markedly abnormal growth properties. When the retroviral vector containing beta-gal without ras (Gal-509) was transferred into DEN-treated rats, 2.5% of the cells were present in clusters containing at least twice as many cells as the largest cluster in Gal-509-transduced, DEN-untreated animals. Thus, p21-ras may increase the percentage of cells that acquire mutations in response to DEN, or it may behave synergistically with other mutations to increase the replication rate of cells. Occasional foci in Ras-gal-transduced, DEN-treated rats had extramedullary hematopoiesis. Forty % of the Ras-gal-transduced, DEN-treated rats developed unifocal HCC, mixed HCC/cholangiocarcinoma (CC), or CC at 3-6 months after transduction, suggesting that hepatocytes can develop into HCC or CC if sufficient genetic alterations occur.

Animals↗

PubMed1995

Experiments in transgenic mice show that hepatocytes are the source for postnatal liver growth and do not stream.

One hypothesis is that postnatal liver growth involves replication of mature hepatocytes, which have an unlimited proliferative potential. An alternative viewpoint is that only certain periportal cells can replicate extensively and that daughter cells stream slowly from the periportal to the pericentral region of the liver. Transgenic mice expressing the beta-galactosidase (beta-gal) gene from the human alpha 1 antitrypsin promoter were used to examine the proliferative potential of hepatocytes. Surprisingly, only 10% of hepatocytes in two different transgenic lines stain blue with X-gal. In neonatal animals, singlets or doublets of expressing cells are randomly scattered throughout the liver. Although the overall frequency of blue cells is similar in older animals, these cells are present in much larger clusters, suggesting that individual expressing cells have replicated to form a clonally derived cluster. Expression patterns are not altered by the administration of an acute phase stimulus or by the performance a partial hepatectomy, suggesting that the expression state cannot be easily altered, and making it more likely that the expression state is indeed fixed. These results suggest that the clusters of blue cells are clonally derived in the transgenic mice. They argue that the parenchymal hepatocyte is responsible for growth in the postnatal liver and that streaming of liver cells does not occur.

Acute-Phase Proteins↗

PubMed1994

Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes.

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347-bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596-bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35-, 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.

Animals↗