Intraportal Injection Research Articles

Human serum albumin microspheres approximate initial organ-specific biodistributions of transplanted hepatocytes and are effective cell surrogates for safety studies.

Liver repopulation with transplanted hepatocytes will generate novel cell-based therapies, although translocation of transplanted cells into lungs through portasystemic shunts has the potential for embolic complications. To facilitate safety analysis of hepatocyte transplantation, we wished to obtain effective cell surrogates and analyzed biodistributions of similarly sized 99mTc-labeled human serum albumin microspheres and rat hepatocytes. Image analysis with dual 99mTc and 111In labels indicated that cells and microspheres were similarly distributed in the liver when injected into normal rats via the spleen. Also, their distributions were similar when injected via a femoral vein or the superior mesenteric vein with cells and microspheres localizing in lungs or liver, respectively. Upon intraportal injection in rats with portal hypertension, microspheres localized in both liver and lungs, consistent with portasystemic shunting. These data demonstrate that human serum albumin microspheres are effective cell surrogates for approximating the safety of hepatocyte transplantation and should be clinically useful.

Effect of MTP on TNF-α in Perfused Rat Liver after Bacteremia and Ischemia/Reperfusion

Introduction.Muramlytripeptide phosphatidylethanolamine (MTP) stimulates synthesis of cytokines by hepatic Kupffer cells. We have shown in a perfused rat liver model that secondary ischemia/reperfusion (I/R) downregulates tumor necrosis factor alpha (TNF-α) expression afterEscherichia coli(EC) bacteremia. Here, we tested the hypothesis that pretreatment with MTP restores cytokine response after sequential bacteremia and I/R.Methods.Thirty-eight livers were studied in eight groups after intraportal injection of 109live EC or normal saline (NS): (1) normoxic EC; (2) EC + I/R (ischemia began 30 min after EC followed by 2 h of reperfusion); (3) normoxic NS controls; and (4) NS + I/R. Four groups of rats received 300 μg/kg of MTP i.v. 24 h prior to liver harvesting: (5) MTP + EC; (6) MTP + EC + I/R; (7) MTP + NS; and (8) MTP + NS + I/R. Bioactive and antigenic TNF-α, PGE2and bacterial clearance were assessed.Results.MTP increased bioactive TNF-α response to EC (MTP + EC vs EC controls: 685 ± 255 U/ml vs 250 ± 180 U/ml,P< 0.02). I/R did not downregulate TNF-α in animals treated with MTP (MTP + NS vs MTP + NS + I/R,P= 0.83). Pretreatment with MTP restored TNF-α after I/R MTP + EC + I/R vs EC + I/R: 671 ± 215 U/ml vs 27 ± 14 U/ml,P< 0.001) to levels similar to those found in the MTP + EC group (MTP + EC + I/R vs MTP + EC: 671 ± 215 U/ml vs 685 ± 255 U/ml,P= 0.75). Finally, bacterial clearance was increased in groups which received MTP.Conclusion. In vivoadministration of MTP increases hepatic TNF-α response to intraportal EC bacteremia by a PGE2independent mechanism. This response is maintained even after subsequent ischemia and reperfusion.

Effect of Hepatic Portal Injection of Ouabain on the Hepato-Sympathoadrenal Reflex

The purpose of the present investigation was to evaluate the effects of an intraportal injection of ouabain (2 mg/kg), an inhibitor of the sodium-potassium pump, on plasma catecholamine response in unrestrained normally fed rats with and without an intact hepatic vagus nerve. Three groups of rats were submitted to two injection conditions each. Hepatic vagotomized (HV) rats were randomly injected with ouabain or saline (0.9%) in the portal vein. Sham-operated rats were either injected with ouabain or saline in the portal or jugular vein. Ouabain or saline were injected at 0 min and again at 20 min. Plasma catecholamines were measured before the first injection and 15 min after each injection. Blood glucose concentrations were significantly ( p < 0.01) increased by the ouabain injection as compared with basal values and saline-injected groups. The hyperglycemic effect of ouabain was not affected by the hepatic vagotomy or the site of infusion. The injection of ouabain, either into the portal or the jugular vein and either after HV or the sham operation, resulted in a significant ( p < 0.01) increase in epinephrine levels as compared with saline-infused rats. Plasma norepinephrine levels were significantly ( p < 0.05) increased after the second intraportal injection of ouabain in both HV and sham-operated groups. However, the injection of ouabain into the jugular vein did not change the plasma norepinephrine levels. The latter observation indicates a specific action of ouabain in the liver on the sympathetic activity.

A new pseudo-peptide analogue of the Arg-Gly-Asp (RGD) sequence inhibits liver metastasis of colon 26-L5 carcinoma cells

We have investigated the effect of the pseudo-peptide analogue (FC-336) of the Arg-Gly-Asp (RGD) sequence in a liver metastasis model by the inoculation of a highly liver-metastatic cell line of colon 26 carcinoma (colon 26-L5) into the portal vein of BALB/c mice. The intraportal injection of colon 26-L5 cells with FC-336 resulted in a marked suppression of liver metastatic colonies in a dose-dependent manner and it reduced the liver weights to a normal level. However, the co-injection of tumor cells with a high dose of RGDS tetrapeptide led to a slight inhibition of liver metastasis. The multiple i.v. administration of FC-336 after tumor inoculation as well as the injection of FC-336 with tumor cells caused significant inhibition of experimental metastasis in the liver. The multiple i.v. administration of the RGDS peptide did not show any inhibitory activity. FC-336 significantly enhanced the survival rate of mice compared with untreated controls when injected intraportally with tumor cells or when intravenously administered after tumor inoculation. Zymography analysis showed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by colon 26-L5 cells, while RGDS peptide did not affect the enzymatic degradation. These findings clearly indicate that the pseudo-peptides of the RGD sequence (FC-336) have a potent inhibitory activity on liver metastasis of colon 26-L5 carcinoma cells.

Improved gene delivery formulations and expression systems for enhanced transaction efficiency

Abstract

A novel method for measuring the hepatic first-pass effect and metabolic rate of L-3,4-dihydroxyphenylalanine (DOPA), diazepam and inulin in rat liver.

A method to analyze in vivo metabolites in blood obtained from the hepatic vein after the intraportal injection of a drug was established. This method includes cannulation into the portal vein, hepatic vein and bile duct, followed by TLC-autoradioluminography of a non-extracted sample. Either [14C]diazepam, L-3,4-dihydroxyphenyL[3-(14)C]alanine or [14C]inulin was administered on the day following the surgical operation to avoid the influence of anesthesia. Radioactive concentrations of [14C]DOPA decreased rapidly in the first 1 min, then decreased gradually. The concentrations of [14C]diazepam increased within the first 1 min and then decreased gradually. The concentration of [14C]inulin was approximately 10 times higher than that of [14C]DOPA, and both decreased gradually. These results show that the uptake levels of the drugs to the liver varied depending on the drugs. The metabolites of [14C]diazepam and [14C]DOPA were detected as early as 5 s after administration. These results suggest that the in vivo hepatic first pass effect of drugs in the early period of injection (5 s) can be studied using these techniques.

A new rat model of portal hypertension induced by intraportal injection of microspheres.

AIM:To produce a new rat model of portal hypertension by intraportal injection of microspheres.METHODS: Measured aliquots of single or different-sized microspheres (15,40,80&mgr;m) were injected into the portal vein to block intrahepatic portal radicals. The resultant changes in arterial,portal,hepatic venous and splenic pulp pressures were monitored.The liver and lungs were excised for histological examination.RESULTS: Portal venous pressure was elevated from basal value of 0.89-1.02 kPa to a steady-state of 1.98-3.19 kPa following the sequential injections of single or different-sized microspheres, with a markedly lowered mean arterial pressure. However, a small-dose injection of 80&mgr;m microspheres (1.8X10(5)) produced a steady-state portal venous pressure of 2.53 plus minus 0.17 kPa,and all rats showed normal arterial pressures. In addition, numerous microspheres were found in the lungs in all experimental groups.CONCLUSION: Portal hypertension can be reproduced in rats by intraportal injection of microspheres at a small dose of 80&mgr;m (1.8X105).Intrahepatic portal-systemic shunts probably exist in the normal rat liver.

Anti-metastatic and immunomodulating properties of the water extract from Celosia argentea seeds.

We have investigated the anti-metastatic effect of Celosia argentea seed extracts (CAE), which have traditionally been used as a therapeutic drug for eye and hepatic diseases in China and Japan. Intraperitoneal (i.p.) administration of CAE for 7 d before tumor inoculation significantly inhibited liver metastasis caused by intraportal injection of colon 26-L5 carcinoma cells in a dose-dependent manner. CAE also showed concentration dependent mitogenic activity on BALB/c whole splenocytes, whereas incubation of the non-adherent fraction of splenocytes with CAE did not induce this activity. CAE has the ability to induce interleukin (IL)-12 production from macrophages in vitro. Following i.p. administration of CAE the maximal levels of IL-12 and interferon (IFN)-gamma production in serum were achieved at 2-3 and 6 h, respectively. Experiments using macrophage- or NK cell-deficient mice revealed that CAE-induced IL-12 in serum was not mediated by macrophages and that IFN-gamma production was mainly dependent on natural killer (NK) cells. Since CAE was inactive when the contributions of macrophages were removed in our system, its inhibitory mechanism is likely to be mainly associated with the activation of macrophages to an anti-metastatic state rather than NK cells. CAE administration resulted in increased production of IL-2, IFN-gamma and decreased production of a Th2 cytokine (IL-4) from splenocytes stimulated by PMA and A23187. Thus, the anti-metastatic effect by CAE is based on its immunomodulating properties including induction of cytokines such as IL-12, IL-2 and IFN-gamma leading to a Th1 dominant immune state and activating macrophages to the tumoricidal state. This may provide a basis for the inhibition of cancer metastasis.

ADENOVIRUS-MEDIATED GENE TRANSFER IN RAT LIVER OF INTERLEUKIN 4 BUT NOT INTERLEUKIN 10 PRODUCES SEVERE ACUTE HEPATITIS

Several immune responses are either limited to or concentrated in a given organ. Cytokines produced during ongoing immune responses have organ-localized effects that can be only partially mimicked upon their systemic delivery. Recombinant adenoviruses are efficient vectors to induce transient organ-localized cytokine expression. This allows in vivo analysis of the effects of cytokines produced spatially and temporally in a manner comparable to that observed during immune responses. The authors generated recombinant adenovirus for rat IL-4 (AdIL-4) and IL-10 (AdIL-10) to analyse the in vivo effects of these two important immunoregulatory molecules after gene transfer in the liver. It was first established that AdIL-4 and AdIL-10 were able to direct the production of biologically active cytokines by different rat cell types in vitro. Intraportal injection of doses of up to 10 10pfu of AdIL-10 or control non-coding recombinant adenovirus were well tolerated, and hepatic histology showed only mild alterations. Conversely, animals receiving more than 2.5×10 9pfu of AdIL-4 showed dose-dependent mortality, with clinical signs of hepatic dysfunction. Liver histology in animals receiving 2.5×10 9pfu of AdIL-4 showed severe acute hepatitis with maximal lesions between day 7 and 14 and almost complete normalization by day 28 after gene transfer. The leukocyte infiltrate was composed primarily of mononuclear cells, but eosinophils and mast cells were significantly increased as compared to control animals. Hepatic function was also altered in animals that received AdIL-4, with kinetics similar to that of histological lesions. Our study describes a model for investigating cytokine function in vivo through liver-localized transgene expression mediated by adenoviral vectors and demonstrates that liver production of IL-4 but not IL-10 results in acute severe hepatitis.

Liver metastases from colorectal cancer: drug delivery with liposome-encapsulated doxorubicin.

To evaluate the intratumoral distribution of liposome-encapsulated doxorubicin. Tumor-bearing livers of 24 mice were studied with in vivo fluorescence and electron microscopy after injection of liposomal doxorubicin in the hepatic artery, portal vein, or tail vein. Distribution and uptake of liposomes and doxorubicin in tumors were compared at 5, 30, and 60 minutes after injection. In vitro evaluation of uptake of doxorubicin in Kupffer cells and in human colorectal cancer cells incubated under normoxic and hypoxic conditions for 5, 30, and 60 minutes was performed with fluorescence microscopy. Doxorubicin autofluorescence was seen in tumors 30 minutes after intraarterial and intraportal injection and was statistically significantly greater at 60 minutes (P < .001). Liposomes were observed in small tumors (diameter < 300 microns) and were trapped in Kupffer cells around larger, hypovascular tumors. Electron microscopy findings confirmed intracytoplasmic, perinuclear uptake of liposomes in tumor cells. In vitro, a higher proportion of doxorubicin was seen in cancer cells (92%) than in Kupffer cells (75%) after 60 minutes incubation. Liposomal doxorubicin can be reliably delivered to liver metastases via the hepatic artery, eliminating need for tumor embolization. Further evaluation is warranted, and the drug may be useful for treating patients with unresectable liver metastases.

Microchimerism and hyporesponsiveness induced by intraportal injection of donor spleen cells in rats

It is controversial whether or not microchimerism (MC) is responsible for the induction and maintenance of donor-specific tolerance. We have shown that intraportal injection (i.p.) of donor splenocytes induces a long-term graft survival of liver and heart in rats. In this study, we examined by polymerase chain reaction (PCR) the status of MC in the liver, spleen, and blood of rat cardiac recipients following i.p. or intravenous injection (i.v.) of donor splenocytes. Male DA (RT1 a) and Wistar (RT1 k) rats were used as donors and recipients, respectively. Heterotopic heart transplantation was performed 10 days after i.p. or i.v. injection of 5 × 10 7 DA spleen cells. DA cardiac allografts were rejected with a mean survival time (MST) of 11.9 ± 1.6 ( n = 10) days in nontreated recipients. Injections (i.v.) led to no significant prolongation of graft survival (MST: 11.2 ± 1.9 days, n = 6), while i.p. injection induced a significant prolongation of graft survival (MST: 18.6 ± 1.5 days, n = 5) over the control. Either i.p. or i.v. injection alone resulted in systemic MC in these organs throughout the observation time over 60 days. MC was detected in the spleen, liver, and blood of cardiac recipients 7 days after transplantation and also even after cessation of cardiac heartbeat 21 days after transplantation. This was the case with either i.p. or i.v. group, which showed MC on day 7 after transplantation and persistent MC after cessation of the heartbeat. These data suggest that the presence of MC in the liver, spleen, and the blood of transplant recipients may not be responsible for immunological unresponsiveness to donor antigens.

Effect of platelet-activating factor on hepatic capillary pressure in isolated dog liver

We determined the effects of platelet-activating factor (PAF), a potent vasoactive autacoid phospholipid, on the capillary pressure and liver weight (Wt) in isolated canine livers perfused with blood bivascularly via the portal vein and hepatic artery. PAF (0.01–33 μg) administered intraportally produced dose-dependent increases in the hepatic capillary pressure, as assessed by triple vascular occlusion pressure (Pto), and Wt. An intraportal injection of 10 μg PAF produced increases in Pto by 10 mmHg and Wt by 35 g 100 g liver weight. This hepatic vasoconstriction was attributed to a threefold increase in the portal vein resistance and a fourfold increase in the hepatic vein resistance. The hepatic arterial resistance was not changed when PAF was arterially or intraportally injected. In conclusion, in isolated perfused dog livers, PAF increases the hepatic capillary pressure and liver weight due to contraction of both the portal vein and hepatic vein, but not the hepatic artery.

Microchimerism and hyporesponsiveness induced by intraportal injection of donor spleen cells in rats.

It is controversial whether or not microchimerism (MC) is responsible for the induction and maintenance of donor-specific tolerance. We have shown that intraportal injection (i.p.) of donor splenocytes induces a long-term graft survival of liver and heart in rats. In this study, we examined by polymerase chain reaction (PCR) the status of MC in the liver, spleen, and blood of rat cardiac recipients following i.p. or intravenous injection (i.v.) of donor splenocytes. Male DA (RT1a) and Wistar (RT1k) rats were used as donors and recipients, respectively. Heterotopic heart transplantation was performed 10 days after i.p. or i.v. injection of 5 x 10(7) DA spleen cells. DA cardiac allografts were rejected with a mean survival time (MST) of 11.9 +/- 1.6 (n = 10) days in nontreated recipients. Injections (i.v.) led to no significant prolongation of graft survival (MST: 11.2 +/- 1.9 days, n = 6), while i.p. or i.v. injection alone resulted in significant MC in these organs throughout the observation time over 60 days. MC was detected in the spleen, liver, and blood of cardiac recipients 7 days after transplantation and also even after cessation of cardiac heartbeat 21 days after transplantation. This was the case with either i.p. or i.v. group, which showed MC on day 7 after transplantation and persistent MC after cessation of the heartbeat. These data suggests that the presence of MC in the liver, spleen and blood of transplant recipients may not be responsible for immunological unresponsiveness to donor antigens.

In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes.

A novel LPD formulation has been developed for in vivo gene transfer. It involves the interaction of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complexes, LPD offers better protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. When a luciferase reporter gene was employed, gene expression was found in all tissues examined including lung, heart, spleen, liver and kidney with the highest expression in the lung. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 micrograms per mouse at which concentration approximately 20 ng luciferase protein per milligram extracted tissue protein could be detected in the lung. Increasing the DNA to 100 micrograms per mouse resulted in toxicity and death of the animal. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h and declined thereafter. High expression was also found in the spleen 6 h after injection but dropped very rapidly thereafter. The in vivo gene expression by LPD was dependent upon the route of administration since intraportal injection of LPD led to about a 100-fold decrease in gene expression in the lung as compared with i.v. injection. Using lacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both the lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be nontoxic and only weakly immunogenic in humans, this novel vector may be useful for clinical gene therapy.

Sinusoidal Flow Block after Warm Ischemia in Rats with Diet-Induced Fatty Liver

Donor livers with massive fatty infiltration reportedly are susceptible to ischemia/reperfusion injury after transplantation, which contributes to risk of primary nonfunction. We investigated the effect of warm ischemia and reperfusion on sinusoidal microcirculation in rats with fatty livers from a choline-deficient diet. Rats were subjected to partial hepatic warm ischemia for 30, 60, or 90 min. In a second study, an anti-ICAM-1 monoclonal antibody was injected intraportally 2 min after a 60-min ischemic period. In both studies, injury was assessed by liver histology 6 hr after vascular clamp release and by animal survival. After 30 min of hepatic warm ischemia, almost all control and fatty-liver rats survived 7 days. After 60-min ischemia, however, survival was significantly less in rats with fatty livers than in controls with normal livers (10% vs 90%,P< 0.0001). Histologically, rats with fatty livers showed marked sinusoidal congestion, especially in the midzone of the acinus, while control rats showed no disturbance in microcirculation. In rats with fatty livers treated with intraportal injection of an anti-ICAM-1 antibody, sinusoidal microcirculation was well preserved and the 7-day survival rate after warm ischemia was improved (50% vs no antibody 10%;P= 0.0112). In fatty livers, midzonal sinusoidal flow block occurs after hepatic warm ischemia and reperfusion. Although intraportal injection of an anti-ICAM-1 monoclonal antibody corrected this microcirculatory failure, animal survival was not as good as for controls without fatty livers. These results suggest that both sinusoidal microcirculatory failure and ischemic hepatocellular damage contribute to warm ischemia/reperfusion injury in fatty livers.

A comparison of locoregional depot and systemic preparations of 9-aminocamptothecin for treatment of liver metastases in a rat tumor model: Superior antitumor activity of sustained-release preparation

Locoregional therapy of hepatic-metastatic disease may overcome the limitations of systemic therapy by allowing tumor drug dose intensification without increasing systemic toxicity. This may result in improved efficacy. We have demonstrated that the novel topoisomerase I inhibitor 9-Aminocamptothecin (9-AC) when dissolved in Ethiodol acts as a sustained release preparation, with good antitumor activity. Its benefit as a depot hepatic intraarterial (i.a.) therapy for hepatic metastases was compared to systemic therapy with an aqueous colloidal dispersion (CD) preparation of 9-AC in a rat model, since a lack of demonstrable benefit of the locoregional therapy, would argue against further clinical evaluation. Fisher rats underwent direct intraportal injection of 2 x 10(5) MADB106 adenocarcinoma cells and were treated 6 to 7 days later with: (a) bolus i.a. 9-AC (60 micrograms) in 60 microliters of Ethiodol; (b) bolus i.a. CD/9-AC (60 micrograms) in 200 microliters water; (c) bolus i.a. Ethiodol only (60 microliters), or (d) CD/9-AC (60 micrograms) in 200 microliters water, via a mini-osmotic pump pumping at 1 microliter/hr for 7 days intraperitoneally (i.p.). Livers were harvested 10 to 12 days later, and the number of metastases on the surface were counted blindly. Bolus hepatic i.a. 9-AC/Ethiodol was found to be significantly superior in reducing the number of hepatic metastases, when compared to the aqueous CD preparation administered in the same manner, or by continuous infusion via mini-osmotic pump i.p. (p < 0.01). Systemic therapy was also associated with substantial toxicity. These results suggest that locoregional therapy of hepatic neoplasms with 9-AC/Ethiodol would be associated with clinical efficacy far exceeding that associated with its systemic administration.

Fos induction in rat brain neurons after stimulation of the hepatoportal Na-sensitive mechanism.

Responses of hepatic afferent nerves to intraportal bolus injection of hypertonic solutions were examined in anesthetized rats. Hepatic afferent nerve activity increased in response to an intraportal injection of 0.75 M NaCl or NaHCO3 but did not respond to a similar injection of 1.5 M mannitol, 0.75 M LiCl, or 0.15 M NaCl, implying that nerves in the hepatoportal area are sensitive to increases in Na concentrations and that this leads to stimulation of hepatic afferent nerve activity. To study central activation in response to stimulation of the hepatic Na-sensitive mechanism, c-fos induction was monitored. After electrical stimulation of hepatic afferent nerves, neurons containing Fos-like immunoreactivity (Fos-li) were found in the area postrema, nucleus of the solitary tract, paraventricular hypothalamic nucleus, and supraoptic nucleus at 90 min after stimulation. Induction of Fos-li was also studied after simultaneous infusion of 0.45 M NaCl into the portal vein and distilled water into the inferior vena cava in conscious rats so as to keep the total amount of solution introduced into the systemic circulation isotonic, thus avoiding changes in mean arterial pressure, plasma osmolality, and plasma NaCl concentrations. Fos-li-containing neurons were found in the same regions in which they were found after electrical stimulation. However, few, if any, Fos-li-containing cells were found if the rats were hepatically denervated or if they received an intraportal infusion of hypertonic LiCl or mannitol. These data provide evidence for involvement of the brain stem and forebrain structures in NaCl regulatory functions induced by stimulation of the hepatoportal Na-sensitive mechanism. However, stimulation of the hepatoportal osmosensitive mechanism does not activate these central structures.

Adenovirus-mediated delivery of human kallistatin gene reduces blood pressure of spontaneously hypertensive rats.

Human kallistatin, or human tissue kallikrein-binding protein (HKBP), is a serine proteinase inhibitor (serpin). Transgenic mice overexpressing rat kallikrein-binding protein are hypotensive. To elucidate therapeutic potentials of kallistatin in hypertension, the human kallistatin gene in an adenoviral vector was directly introduced into spontaneously hypertensive rats (SHR) through portal vein injection. The kallistatin cDNA construct (RSV-cHKBP) under the promoter control of Rous sarcoma virus 3' long terminal repeat (LTR) was incorporated into adenovirus (Ad.RSV-cHKBP). Recombinant kallistatin in 293 cells transfected with RSV-cHKBP or Ad.RSV-cHKBP was measured by ELISA and by its complex formation with tissue kallikrein. A single intraportal vein injection of Ad.RSV-cHKBP at a dose of 8 x 10(10) pfu results in a significant reduction of blood pressure of SHR for 4 weeks. Human kallistatin mRNA was detected in the liver, spleen, kidney, aorta, and lung of rats receiving gene delivery. Immunoreactive human kallistatin in rat serum was detected at the highest level 1 day post injection and at lesser amounts in rat tissues. This study shows that adenovirus harboring Ad.RSV-cHKBP produces functional kallistatin, and adenovirus-mediated transfer of the human kallistatin gene reduces blood pressures of SHR. The results suggest that kallistatin may function as a vasodilator in vivo and provide important information for a potential gene therapy approach to hypertension.

Gene transfer and therapy with adenoviral vector in rats with diethylnitrosamine-induced hepatocellular carcinoma.

In rats with diethylnitrosamine (DENA)-induced hepatocellular carcinoma (HCC), we studied in vivo gene transfer efficiency using intraportal injections of recombinant adenovirus carrying the lacZ reporter gene (AdCMVlacZ) and the therapeutic efficacy of adenovirus-mediated transfer of the thymidine kinase gene of the herpes simplex virus (HSV-tk) followed by ganciclovir (GCV) administration. DENA was very effective in inducing HCC but also stimulated nontumor cell replication, as shown by proliferating cell nuclear antigen (PCNA) staining. The study of in vivo gene transfer efficiency in tumor-bearing rats showed that nontumor tissue and small tumor nodules were transduced effectively whereas a poor transduction rate was noted in large tumor nodules. Concerning therapeutic efficacy, three groups of rats with established HCC were studied: group A and B received intraportally recombinant adenovirus carrying HSV-tk (AdCMVtk) or AdCMVlacZ, respectively, and 2 days after GCV was given intraperitoneally for 9 days; group C received only saline. Of the rats from groups B and C, 100% and 93% respectively, exhibited multiple HCC tumor nodules at end of the study. In contrast, a complete regression of tumor was observed in 63% of animals from group A. This group showed significant elevation of serum transaminases and a diffuse hepatotoxic lesion in liver tissue; histological signs of regeneration were observed in surviving animals. Nine out of 19 rats from group A died during the treatment period. We conclude that (i) in the DENA model of HCC, tumoral cells can be destroyed in vivo by the HSV-tk/GCV system despite poor transduction of large tumor nodules, suggesting that toxic metabolites generated by nontumor cells may exert a bystander effect on tumor tissue; (ii) significant hepatoxicity and a high mortality rate occurred in HSV-tk/GCV-treated rats; these side effects appear to be due to the fact that in DENA-treated livers enhanced cell proliferation was present not only in tumor nodules but also in nontumor parenchyma, leading to GCV sensitization of both tissues; (iii) our results have implications concerning the efficacy and potential risks of the HSV-tk/GCV system in the treatment of human HCC.

The action of ATP on the hepatic arterial and portal venous vascular networks of the rabbit liver: the role of adenosine

ATP is released from blood vessels during periods of hypoxia and may be responsible for hepatic arterial vasodilatation during instances of reduced hepatic portal venous flow. The role of adenosine in ATP-induced vasodilator and vasoconstrictor responses of the hepatic arterial and portal venous vascular networks respectively was studied in the isolated dual-perfused rabbit liver in vitro to ascertain whether ATP could be catabolised to adenosine during transit through the hepatic parenchyma. Intra-arterial and intra-portal injections of ATP (−10 to −4 log mol/100 g liver) resulted in dose-dependent vasodilatation in the hepatic artery and vasoconstriction in the portal vein. Addition of 8-phenyltheophylline (10 μM), a non-selective P 1-purinoceptor antagonist, to the hepatic arterial and portal venous perfusate significantly inhibited the hepatic arterial ED 50 for responses to intra-arterial injected ATP from −8.70±0.22 to −7.63±0.28 log mol/100 g liver ( P<0.001); it also inhibited hepatic arterial responses to, mid-range, portal venous injections of ATP. The data suggest that the hepatic arterial vasodilatation to ATP is partly mediated via catabolism to adenosine and may be an important mechanism during periods of relative hepatic hypoxia associated with portal flow reduction.