Preclinical Evaluation of Ureidosulfamate Carbonic Anhydrase IX/XII Inhibitors in the Treatment of Cancers.

Carbonic anhydrases (CAs) are a family of enzymes involved in the pH regulation of metabolically active cells/tissues. Upregulation of the CAIX/XII isoforms is associated with hypoxic tumours and clinically linked with malignant progression, treatment resistance and poor prognosis. The elucidation of the crystal structure of the catalytic domains of CAIX/XII provided the basis for the generation of CAIX/XII selective inhibitors based on the sulfonamide, sulfamate and coumarins chemical structures. Ureido-substituted benzenesulfonamide CAIX/XII inhibitors have shown significant potential, with U-104 (SLC-0111) currently present in clinical Phase I/II. Ureido-substituted sulfamate CAIX/XII inhibitors have received less attention despite encouraging preclinical test results. In triple-negative breast cancer (TNBC), ureidosulfamates revealed a significant antitumour (FC9-398A) and antimetastatic potential (S4). In small cell lung cancer (SCLC), a cancer cell type very sensitive to a dysregulation in CAIX signaling, S4 treatment was particularly effective when combined with cisplatin with no evidence of acquired cisplatin-resistance. These successful anticancer strategies should provide a solid basis for future studies on ureido-substituted sulfamates.

Novel carbonic anhydrase IX-targeted therapy enhances the anti-tumour effects of cisplatin in small cell lung cancer.

Small cell lung cancer (SCLC) has an extremely poor prognosis and methods of improving chemotherapeutic intervention are much sought after. A promising approach lies in inhibiting the tumour-associated enzyme, carbonic anhydrase IX (CA IX), which supports tumour cell survival. The aim of this study was to assess the potential of CA IX inhibition using 4-(3'-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate (S4), for the treatment of human SCLC alone and in combination with cisplatin chemotherapy. Treating SCLC cell lines (DMS 79 and COR-L24) with 100 µM S4 reduced viability in vitro and enhanced cell death when combined with 7 µM cisplatin, most prominently under hypoxic conditions (0.1% O2 ). When either cell line was grown as a xenograft tumour in nude mice, intraperitoneal injection of 50 mg/kg S4 alone and in combination with 3 mg/kg cisplatin led to significantly reduced tumour growth. Combination therapy was superior to single agents and response was greatly accentuated when administering repeated doses of cisplatin in DMS 79 tumours. The mechanism of therapeutic response was investigated in vitro, where S4 treatment increased apoptosis under hypoxic conditions in both DMS 79 and COR-L24 cells. DMS 79 tumours receiving S4 in vivo also displayed increased apoptosis and necrosis. Combining S4 with cisplatin reduced both the area of hypoxia and CA IX-positive cells within tumours and increased necrosis, suggesting hypoxia-specific targeting. This study presents a novel, targeted approach to improving current SCLC therapy via inhibition of CA IX, which enhances apoptosis and significantly inhibits xenograft tumour growth when administered alone and in combination with cisplatin chemotherapy.

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Carbonic anhydrase IX as a target for metastatic disease.

Metastatic disease is responsible for the majority of cancer related deaths. Tumour-associated carbonic anhydrase IX (CA IX) is a powerful marker to diagnose various types of metastatic cancers including those of cervical, renal, breast and head & neck origin. The precise prognostic role of CA IX in determining local control versus overall survival is complex, although the majority of reports favour CA IX as a marker for poor prognosis in patients with metastatic cancer. Preclinical studies in cell cultures clearly demonstrate that CA IX stimulates features that enhance metastatic properties of cancer cells for example, reducing cell adhesion, increasing motility and migration, inducing vascularisation and activating proteases, in which CA IX-induced acidification of the microenvironment of the tumour is essential. As most findings are consistent with the idea that CA IX is important in metastatic dissemination, small molecular CA IX inhibitors (including fluorescent-tagged or radiolabelled) and monoclonal antibodies targeting the CA IX isoform have been developed. Studies in tumour xenograft models showed that these CA IX-specific inhibitors and antibodies can be very effective in therapy and imaging of a variety of different metastatic cancers.

Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cells in vitro.

Carbonic anhydrase IX (CA IX) is a hypoxia-regulated enzyme, overexpressed in many types of human cancer. CA IX is involved in pH homeostasis, contributing to extracellular acidification and tumourigenesis. Acidification of the extracellular milieu can impact upon cellular uptake of chemotherapeutic drugs by favouring weak acids (e.g. melphalan), but limiting access of weak bases (e.g. doxorubicin). We investigated whether alterations of CA IX activity affected anti-cancer drug uptake and toxicity. CA inhibitor acetazolamide (AZM) enhanced doxorubicin toxicity but reduced melphalan toxicity in cell lines that highly expressed CA IX under anoxic conditions (HT29 and MDA435 CA9/18). The toxicity changes reflected modification of passive drug uptake. AZM did not alter toxicity or uptake in cells with low CA IX activity (HCT116 and MDA435 EV1). AZM lowered intracellular pH in HT29 and MDA435 CA9/18 cells under anoxic conditions. CA IX activity has chemomodulatory properties and is an attractive target for anti-cancer therapy.

The c-Rel subunit of nuclear factor-kappaB regulates murine liver inflammation, wound-healing, and hepatocyte proliferation.

UNLABELLED: In this study, we determined the role of the nuclear factor-kappaB (NF-kappaB) subunit c-Rel in liver injury and regeneration. In response to toxic injury of the liver, c-Rel null (c-rel(-/-)) mice displayed a defect in the neutrophilic inflammatory response, associated with impaired induction of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted; also known as CCL5). The subsequent fibrogenic/wound-healing response to both chronic carbon tetrachloride and bile duct ligation induced injury was also impaired and this was associated with deficiencies in the expression of fibrogenic genes, collagen I and alpha-smooth muscle actin, by hepatic stellate cells. We additionally report that c-Rel is required for the normal proliferative regeneration of hepatocytes in response to toxic injury and partial hepatectomy. Absence of c-Rel was associated with blunted and delayed induction of forkhead box M1 (FoxM1) and its downstream targets cyclin B1 and Cdc25C. Furthermore, isolated c-rel(-/-) hepatocytes expressed reduced levels of FoxM1 and a reduced rate of basal and epidermal growth factor-induced DNA synthesis. Chromatin immunoprecipitation revealed that c-Rel binding to the FoxM1 promoter is induced in the regenerating liver. CONCLUSION: c-Rel has multiple functions in the control of liver homeostasis and regeneration and is a transcriptional regulator of FoxM1 and compensatory hepatocyte proliferation.

A Novel Mechanism of Ataxia Telangiectasia Mutated Mediated Regulation of Chromatin Remodeling in Hypoxic Conditions.

The effects of genotoxic stress can be mediated by activation of the Ataxia Telangiectasia Mutated (ATM) kinase, under both DNA damage-dependent (including ionizing radiation), and independent (including hypoxic stress) conditions. ATM activation is complex, and primarily mediated by the lysine acetyltransferase Tip60. Epigenetic changes can regulate this Tip60-dependent activation of ATM, requiring the interaction of Tip60 with tri-methylated histone 3 lysine 9 (H3K9me3). Under hypoxic stress, the role of Tip60 in DNA damage-independent ATM activation is unknown. However, epigenetic changes dependent on the methyltransferase Suv39H1, which generates H3K9me3, have been implicated. Our results demonstrate severe hypoxic stress (0.1% oxygen) caused ATM auto-phosphorylation and activation (pS1981), H3K9me3, and elevated both Suv39H1 and Tip60 protein levels in FTC133 and HCT116 cell lines. Exploring the mechanism of ATM activation under these hypoxic conditions, siRNA-mediated Suv39H1 depletion prevented H3K9me3 induction, and Tip60 inhibition (by TH1834) blocked ATM auto-phosphorylation. While MDM2 (Mouse double minute 2) can target Suv39H1 for degradation, it can be blocked by sirtuin-1 (Sirt1). Under severe hypoxia MDM2 protein levels were unchanged, and Sirt1 levels depleted. SiRNA-mediated depletion of MDM2 revealed MDM2 dependent regulation of Suv39H1 protein stability under these conditions. We describe a novel molecular circuit regulating the heterochromatic state (H3K9me3 positive) under severe hypoxic conditions, showing that severe hypoxia-induced ATM activation maintains H3K9me3 levels by downregulating MDM2 and preventing MDM2-mediated degradation of Suv39H1. This novel mechanism is a potential anti-cancer therapeutic opportunity, which if exploited could target the hypoxic tumor cells known to drive both tumor progression and treatment resistance.

Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated kinase-1.

BACKGROUND: Expressed in embryonic development, matrix metalloprotein-9 (MMP-9) is absent in most of developed adult tissues, but recurs in inflammation during tissue injury, wound healing, tumor formation and metastasis. Expression of MMP-9 is tightly controlled by extracellular cues including pro-inflammatory cytokines and extracellular matrix (ECM). While the pathologic functions of MMP-9 are evident, the intracellular signaling pathways to control its expression are not fully understood. In this study we investigated mechanism of cytokine induced MMP-9 with particular emphasis on the role of p21-activated-kinase-1 (PAK1) and the down stream signaling. RESULTS: In response to TNF-alpha or IL-1alpha, PAK1 was promptly activated, as characterized by a sequential phosphorylation, initiated at threonine-212 followed by at threonine-423 in the activation loop of the kinase, in human skin keratinocytes, dermal fibroblasts, and rat hepatic stellate cells. Ectopic expression of PAK1 variants, but not p38 MAP kinase, impaired the TNF-alpha-induced MMP-9 expression, while other MMPs such as MMP-2, -3 and -14 were not affected. Activation of Jun N-terminal kinase (JNK) and NF-kappaB has been demonstrated to be essential for MMP-9 expression. Expression of inactive PAK1 variants impaired JNK but not NF-kappaB activation, which consequently suppressed the 5'-promoter activities of the MMP-9 gene. After the cytokine-induced phosphorylation, both ectopically expressed and endogenous PAK1 proteins were promptly accumulated even in the condition of suppressing protein synthesis, suggesting the PAK1 protein is stabilized upon TNF-alpha stimulation. Stabilization of PAK1 protein by TNF-alpha treatment is independent of the kinase catalytic activity and p21 GTPase binding capacities. In contrast to epithelial cells, mesenchymal cells require 3-dimensional type-I collagen in response to TNF-alpha to massively express MMP-9. The collagen effect is mediated, in part, by boost JNK activation in a way to cooperate the cytokine signaling. CONCLUSION: We identified a novel mechanism for MMP-9 expression in response to injury signals, which is mediated by PAK1 activation and stabilization leading JNK activation.

The Sulfamate Small Molecule CAIX Inhibitor S4 Modulates Doxorubicin Efficacy.

Carbonic anhydrase IX (CAIX) is a tumor-specific protein that is upregulated during hypoxic conditions where it is involved in maintaining the pH balance. CAIX causes extracellular acidification, thereby limiting the uptake of weak basic chemotherapeutic agents, such as doxorubicin, and decreasing its efficacy. The aim of this study was to determine if doxorubicin efficacy can be increased when combined with the selective sulfamate CAIX inhibitor S4. The effect of S4 on doxorubicin efficacy was tested in vitro using cell viability assays with MDA-MB-231, FaDu, HT29 -CAIX high and HT29 -CAIX low cell lines. In addition, the efficacy of this combination therapy was investigated in tumor xenografts of the same cell lines. The addition of S4 in vitro increased the efficacy of doxorubicin in the MDA-MB-231 during hypoxic exposure (IC50 is 0.25 versus 0.14 µM, p = 0.0003). Similar results were observed for HT29-CAIX high with S4 during normoxia (IC50 is 0.20 versus 0.08 µM, p<0.0001) and in the HT29 -CAIX low cells (IC50 is 0.09 µM, p<0.0001). In vivo doxorubicin treatment was only effective in the MDA-MB-231 xenografts, but the efficacy of doxorubicin was decreased when combined with S4. In conclusion, the efficacy of doxorubicin treatment can be increased when combined with the selective sulfamate CAIX inhibitor S4 in vitro in certain cell lines. Nevertheless, in xenografts S4 did not enhance doxorubicin efficacy in the FaDu and HT29 tumor models and decreased doxorubicin efficacy in the MDA-MB-231 tumor model. These results stress the importance of better understanding the role of CAIX inhibitors in intratumoral pH regulation before combining them with standard treatment modalities, such as doxorubicin.

Dissemination via the lymphatic or angiogenic route impacts the pathology, microenvironment and hypoxia-related drug response of lung metastases.

Complications associated with the development of lung metastases have a detrimental effect on the overall survival rate of many cancer patients. Preclinical models that mimic the clinical aspects of lung metastases are an important tool in developing new therapy options for these patients. The commonly used intravenous models only recapitulate dissemination of cancer cells to the lungs via the haematological route. Here we compared spontaneous and intravenous lung metastases of the highly metastatic KHT mouse fibrosarcoma cells after injecting KHT cells into the subcutaneous layer of the skin or directly into the tail vein. In contrast to the intravenous model, metastases spontaneously arising from the subcutaneous tumours disseminated most consistent with the lymph nodes/lymphatics route and were more hypoxic than the metastases observed following tail-vein administration and haematological spread. To ascertain whether this impacted on drug response, we tested the effectiveness of the hypoxia-sensitive cytotoxin AQ4N (Banoxantrone) in both models. AQ4N was more effective as an anti-metastatic drug in mice with subcutaneous KHT tumours, significantly reducing the metastatic score. Complementing the KHT studies, pathology studies in additional models of spontaneous lung metastases showed haematological (HCT116 intrasplenic implant) or mixed haematological/lymphatic (B16 intradermal implant) spread. These data suggest that preclinical models can demonstrate differing, clinically relevant dissemination patterns, and that careful selection of preclinical models is required when evaluating new strategies for targeting metastatic disease.

[18F]-FLT positron emission tomography can be used to image the response of sensitive tumors to PI3-kinase inhibition with the novel agent GDC-0941.

The phosphoinositide 3-kinase (PI3K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the positron emission tomography tracer 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]-FLT) is suitable to mark the effect of the novel PI3K inhibitor GDC-0941, which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [(18)F]-FLT and at acute (18 hours) and chronic (186 hours) time points after twice-daily administration of GDC-0941 (50 mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [(18)F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3K p85 subunit p85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [(18)F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute time point and correlated with pharmacodynamic markers of PI3K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3K signaling via expression of Δp85α significantly reduced tumor growth and [(18)F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [(18)F]-FLT is a strong candidate for the noninvasive measurement of GDC-0941 action.

Antimetastatic effect of sulfamate carbonic anhydrase IX inhibitors in breast carcinoma xenografts.

A panel of compounds belonging to the underexposed sulfamate class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors was generated that displayed high specificity at nanomolar levels for the tumor-associated CA IX/XII isoforms. Three of the specific CA IX/XII inhibitors showed a positive response in in vitro assays for tumor cell migration and spreading. One of them, 4-(3'-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate (S4), was taken forward into the orthotopic MDA-MB-231 (breast carcinoma) model in mice. Treatment with a 10 mg/kg maintenance dosage of S4 given daily on a "5 days on, 2 days off" regimen reduced metastatic tumor burden in the lung while not affecting primary tumor growth or mouse condition. CA inhibitors of the sulfamate class specifically targeting the tumor-associated isoforms are potential candidates in antimetastatic therapy.

Interleukin-1 participates in the progression from liver injury to fibrosis.

Interleukin-1 (IL-1) is rapidly expressed in response to tissue damage; however, its role in coordinating the progression from injury to fibrogenesis is not fully understood. Liver fibrosis is a consequence of the activation of hepatic stellate cells (HSCs), which reside within the extracellular matrix (ECM) of subsinusoids. We have hypothesized that, among the hepatic inflammatory cytokines, IL-1 may directly activate HSCs through autocrine signaling and stimulate the matrix metalloproteinases (MMPs) produced by HSCs within the space of Disse, resulting in liver fibrogenesis. In this study, we first established a temporal relationship between IL-1, MMPs, HSC activation, and early fibrosis. The roles of IL-1 and MMP-9 in HSC activation and fibrogenesis were determined by mice deficient of these genes. After liver injury, IL-1, MMP-9, and MMP-13 levels were found to be elevated before the onset of HSC activation and fibrogenesis. IL-1 receptor-deficient mice exhibited ameliorated liver damage and reduced fibrogenesis. Similarly, advanced fibrosis, as determined by type-I and -III collagen mRNA expression and fibrotic septa, was partially attenuated by the deficiency of IL-1. In the early phase of liver injury, the MMP-9, MMP-13, and TIMP-1 expression correlated well with IL-1 levels. In injured livers, MMP-9 was predominantly colocalized to desmin-positive cells, suggesting that HSCs are MMP-producing cells in vivo. MMP-9-deficient mice were partially protected from liver injury and HSC activation. Thus IL-1 is an important participant, along with other cytokines, and controls the progression from liver injury to fibrogenesis through activation of HSCs in vivo.

Fibrosis and cirrhosis reversibility - molecular mechanisms.

The concept that liver fibrosis is a dynamic process with potential for regression as well as progression has emerged in parallel with clinical evidence for remodeling of fibrotic extracellular matrix in patients who can be effectively treated for their underlying cause of liver disease. This article reviews recent discoveries relating to the cellular and molecular mechanisms that regulate fibrosis regression, with emphasis on studies that have used experimental in vivo models of liver disease. Apoptosis of hepatic myofibroblasts is discussed. The functions played by transcription factors, receptor-ligand interactions, and cell-matrix interactions as regulators of the lifespan of hepatic myofibroblasts are considered, as are the therapeutic opportunities for modulating these functions. Growth factors, proteolytic enzymes, and their inhibitors are discussed in detail.

NF-kappaB is a critical regulator of the survival of rodent and human hepatic myofibroblasts.

BACKGROUND/AIMS: Hepatic myofibroblast activation during injury causes deposition of extracellular matrix within the liver and promotes development of fibrosis. Hepatic myofibroblast apoptosis is associated with remodelling of fibrotic extracellular matrix and regression of fibrosis. Previous work showed that inhibition of constitutive NF-kappaB signaling promotes hepatic myofibroblast apoptosis and resolution of fibrosis in rodent models. However, to date agents used to target constitutive NF-kappaB transcriptional activity in hepatic myofibroblasts have been relatively non-specific with potential for off-target effects that may complicate data interpretation. Likewise, rat chronic liver disease models may not accurately recapitulate the activation of human hepatic myofibroblasts. METHODS: We used a mutant recombinant IkappaBalpha super-repressor fused to the HIV-TAT domain to specifically target NF-kappaB signaling in hepatic myofibroblasts. Inhibition of NF-kappaB activity was measured using reporter assay. Apoptosis of hepatic myofibroblasts was assessed by morphological changes, cleavage of the PARP-1 protein and Caspase 3 activation. RESULTS: TAT-IkappaBalphaSR reduced NF-kappaB dependent transcription, Bcl-2 expression and promoted Jun-N-terminal kinase-dependent apoptosis in human and rat hepatic myofibroblasts. CONCLUSIONS: These data highlight the conserved role of NF-kappaB during fibrogenesis. Our data validate the use of rodent models for pre-clinical testing of NF-kappaB inhibitors as anti-fibrotics and stimulators of fibrotic extracellular matrix remodelling.

Stereological measurement of porto-central gradients in gene expression in mouse liver.

The liver is thought to consist of lobules, numerous repeating, randomly oriented units. Within these lobules, genes are expressed in gradients along the porto-central axis, which spans the distance between portal and central veins. We have developed a robust stereological method to map all points in an image to their position on this porto-central axis. This approach is based on the distribution of well-characterized periportal and pericentral enzymes, which are visualized on sections preceding and following the section of interest. Because expression of the model genes phosphoenolpyruvate carboxykinase and ornithine aminotransferase declines gradually with increasing distance from the portal vein and central vein, respectively, these genes can be used to prepare images with topographical information without any assumption about the shape of the hepatic unit, or about the direction or shape of the gradient to be determined. The "relative distance" image is a 2-dimensional image that accurately maps the relative position of hepatocytes on the porto-central axis in 3-dimensional space. It is superimposed on the serial section under investigation to relate local staining density to position on the porto-central axis and obtain the gene expression gradient. The method was used to determine the expression gradient of 2 periportal and 2 pericentral enzymes and their response to fasting. The "total distance" image was used to measure the length of the porto-central axis, which was approximately 210 microm in mice and found to decrease 13% after 1 day of starvation. The method can be applied to any tissue component that can be stained quantitatively.

Hepatic response to right ventricular pressure overload.

BACKGROUND & AIMS: Modifying the afferent blood supply to the liver does not change the zonal expression pattern of hepatic enzymes in the rat. METHODS: We used pulmonary trunk banding (PTB) to study the effect of an efferent hindrance of blood flow on hepatic architecture and zonation of gene expression. RESULTS: Most PTB rats developed right ventricular hypertrophy and congestive heart failure. The hepatic response to PTB developed concomitantly with the decline in heart function. Enzyme expression in the periportal region was not affected, but the pericentral rim of hepatocytes expressing glutamine synthetase, ornithine aminotransferase, and NADPH cytochrome P-450 reductase (CYPred) first declined in diameter, then became discontinuous, and finally disappeared. Meanwhile, ornithine aminotransferase and especially CYPred, became re-expressed in the periportal zone. These changes occurred without appreciable cell death or fibrotic changes; the expression of fibronectin and alpha-smooth muscle actin increased perisinusoidally, but that of collagen did not. Electron microscopic analysis revealed normal fenestration of the sinusoidal endothelial cells without detectable deposition of basement membrane material, but both the width of the space of Disse and the length and number of hepatic microvilli were significantly reduced, implying a decreased flow of fluid in the space of Disse. CONCLUSIONS: The reprogramming of gene expression in livers with a postsinusoidal hindrance of blood flow results from declining access of the hepatocytes to intrasinusoidal signal-transduction molecules and suggest that the impaired biotransformation that accompanies right ventricular failure is caused by a central-to-portal shift in expression of the corresponding enzymes.