Differential regulation of rodent hepatocyte and oval cell proliferation by interferon gamma

Hepatocytes and intrahepatic progenitor cells (oval cells) have similar responses to most growth factors but rarely proliferate together. Oval cells constitute a reserve compartment that is activated when hepatocyte proliferation is inhibited. Interferon gamma (IFN-gamma) increases in liver injury that involves oval cell responses, but it is not upregulated during liver regeneration after partial hepatectomy. Based on these observations, we used well-characterized lines of hepatocytes (AML-12 cells) and oval cells (LE-6 cells) to investigate the potential mechanisms that regulate differential growth responses in hepatocytes and oval cells. We show that IFN-gamma blocks hepatocyte proliferation in vivo, and that in combination with either tumor necrosis factor (TNF) or lipopolysaccharide (LPS), it causes cell cycle arrest in hepatocytes but stimulates oval cell proliferation in cultured cells. The hepatocyte cell cycle arrest is reversible, is p53-independent, and is not associated with apoptosis. Treatment of AML-12 hepatocytes with IFN-gamma/LPS or IFN-gamma/TNF, but not with individual cytokines, induced NO synthase and generated NO, while similarly treated oval cells produced little if any NO. Generation of NO by an NO donor reproduced the inhibitory effect of the cytokine combinations on AML-12 cell replication, while NO inhibitors abolish the replication deficiency. In conclusion, we propose that IFN-gamma, in conjunction with TNF or LPS, can both inhibit hepatocyte proliferation through the generation of NO and stimulate oval cell replication. The response of hepatocytes and oval cells to cytokine combinations may contribute to the differential proliferation of these cells in hepatic growth processes.     

Transforming growth factor beta mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation.

Transforming growth factor beta (TGF-beta) is a growth factor with multiple biological properties including stimulation and inhibition of cell proliferation. To determine whether TGF-beta is involved in hepatocyte growth responses in vivo, we measured the levels of TGF-beta mRNA in normal liver and during liver regeneration after partial hepatectomy in rats. TGF-beta mRNA increases in the regenerating liver and reaches a peak (about 8 times higher than basal levels) after the major wave of hepatocyte cell division and mitosis have taken place and after the peak expression of the ras protooncogenes. Although hepatocytes from normal and regenerating liver respond to TGF-beta, they do not synthesize TGF-beta mRNA. Instead, the message is present in liver nonparenchymal cells and is particularly abundant in cell fractions enriched for endothelial cells. TGF-beta inhibits epidermal growth factor-induced DNA synthesis in vitro in hepatocytes from normal or regenerating liver, although the dose-response curves vary according to the culture medium used. We conclude that TGF-beta may function as the effector of an inhibitory paracrine loop that is activated during liver regeneration, perhaps to prevent uncontrolled hepatocyte proliferation.     

Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice

Transforming growth factor beta (TGF-beta) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF-beta signaling in liver regeneration in vivo, the TGF-beta type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing "floxed" Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF-beta1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF-beta regulates G(1) to S phase transition of hepatocytes, but intact signaling by TGF-beta is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF-beta pathway is abolished, and may be a principal factor in the termination of liver regeneration.     

Different effects of angiogenesis inhibitors IFN-alpha and TIMP-1 on lymphangiogenesis

This study was designed to examine the effects of angiogenesis inhibitors IFN-alpha and TIMP-1 on lymphangiogenesis. We cultured lymphatic endothelial (LE) cells from pig thoracic ducts and performed morphological observations using light microscopy, TEM, and confocal microscopy to confirm their lymphatic origin. We tested these cells for growth inhibition by angiogenesis inhibitors IFN-alpha and TIMP-1 using both the scraping line and MTT methods. In addition, we analyzed apoptosis using the Hoechst and Caspase staining methods. Finally, we tested IFN-alpha and TIMP-1 using in vivo inhibitory assays. By morphological observations, all LE cells in vivo and in vitro were found to be of very similar morphology. Both in vitro inhibitory assays of scraping line and MTT showed significant differences for the IFN-alpha treatment (p < 0.01) and no significant difference for TIMP-1. Hoechst and Caspase apoptosis assays demonstrated that IFN-alpha could induce apoptosis of LE cells, and TIMP-1 had little effect. IFN-alpha and TIMP-1 inhibitory in vivo assays showed a lack of healing following IFN-alpha treatment compared to control and TIMP-1 treatment. In summary, these different angiogenesis inhibitors have different effects on lymphangiogenesis. IFN-alpha inhibits proliferation and migration of LE cells in a dose-dependent fashion and induces apoptosis of LE cells while TIMP-1 has no significant inhibitory effects on proliferation, migration, or inducing apoptosis.     

TGF-beta/Smad signaling in the injured liver

TGF-beta, acting both directly and indirectly, represents a central mediator of fibrogenic remodeling processes in the liver. Besides hepatic stellate cells (HSCs), which are induced by TGF-beta to transdifferentiate to myofibroblasts and to produce extracellular matrix, hepatocytes are also strongly responsive for this cytokine, which induces apoptosis during fibrogenesis and provides growth control in regeneration processes. Based on this, TGF-beta-mediated hepatic responses to injury are the result of a complex interplay between the different liver cell types. In this review we summarize the knowledge about TGF-beta signal transduction in HSCs with special impact on Smad pathways. We further describe a molecular cross-talk between profibrogenic TGF-beta and antifibrogenic IFN-gamma signaling in liver cells. Finally, we introduce hepatocyte plasticity and epithelial-to-mesenchymal transition in the liver, which is well established in tumorigenesis, as a potential feature of fibrogenesis and highlight possible action points of TGF-beta in these contexts.     

TGF-beta signaling-deficient hematopoietic stem cells have normal self-renewal and regenerative ability in vivo despite increased proliferative capacity in vitro

Studies in vitro implicate transforming growth factor beta (TGF-beta) as a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. In vivo studies have been hampered by early lethality of knock-out mice for TGF-beta isoforms and the receptors. To directly assess the role of TGF-beta signaling for hematopoiesis and hematopoietic stem cell (HSC) function in vivo, we generated a conditional knock-out model in which a disruption of the TGF-beta type I receptor (T beta RI) gene was induced in adult mice. HSCs from induced mice showed increased proliferation recruitment when cultured as single cells under low stimulatory conditions in vitro, consistent with an inhibitory role of TGF-beta in HSC proliferation. However, induced T beta RI null mice show normal in vivo hematopoiesis with normal numbers and differentiation ability of hematopoietic progenitor cells. Furthermore HSCs from T beta RI null mice exhibit a normal cell cycle distribution and do not differ in their ability long term to repopulate primary and secondary recipient mice following bone marrow transplantation. These findings challenge the classical view that TGF-beta is an essential negative regulator of hematopoietic stem cells under physiologic conditions in vivo.     

Transforming growth factor-beta and hepatocyte transdifferentiation in liver fibrogenesis

Currently, hepatic stellate cells (HSC) are thought to be the major fibrotic precursor cells that transdifferentiate to fibrogenic, extracellular matrix producing myofibroblasts in inflammatory liver tissue upon transforming growth factor-beta (TGF-beta) signaling, whereas hepatocytes are thought to respond with apoptosis to this cytokine. Starting out from in vitro experiments with primary hepatocyte cultures and immortalized AML-12 cells, TGF-beta signaling in this cell type was assessed and apoptosis was found to be only a minor effect. Instead, hepatocytes undergo epithelial mesenchymal transition (EMT), a physiological process in embryogenesis and of relevance for cancerous cell transformation. In injured liver, however, this process contributes to the promotion of fibrosis. Already after a few days of culture, hepatocytes lose their epithelial honeycomb-like shape towards a fibroblast-like phenotype. We could demonstrate by microarray analysis that stimulation of hepatocytes with TGF-beta regulates the expression of genes involved in EMT and fibrosis. Among these were, for example, Snail, a known mediator of EMT, and connective tissue growth factor (CTGF), a strong inducer of fibrosis. In a mouse model, hepatocyte-specific overexpression of Smad7 was able to blunt a fibrogenic response after CCl(4) intoxication. These results emphasize the dynamic nature of liver fibrosis, challenge the paradigm of HSC as a crucial source of liver myofibroblasts and hint towards a prominent role for hepatocytes in liver fibrogenesis.     

Transformed fat storing cells inhibit the proliferation of cultured hepatocytes by secretion of transforming growth factor beta

Conditioned medium from secondary cultures of fat storing cells (transformed fat storing cells) inhibits replicative (hydroxyurea-sensitive) DNA synthesis dose-dependently in primary cultures of hepatocytes stimulated in vitro by transforming growth factor (TGF) alpha. Similarly, [3H]thymidine incorporation into the DNA of hepatocytes from the regenerating rat liver is reduced by about 70% by fat storing, cell conditioned medium. Medium which had been transiently acidified was more potent than native medium. It displaced [125I]TGF-beta from binding sites on the hepatocyte surface and the inhibitory activity was completely blocked by anti-TGF-beta antibodies. From binding studies, a TGF-beta concentration of 1.8 +/- 0.4 ng/ml conditioned medium produced by 2 X 10(5) cells per 24 h was estimated. Transformed, but not primary, cultures of fat storing cells at an early state produce and secrete TGF-beta, which reduces hepatocellular proliferation significantly.     

Ductular proliferation in liver tissues with severe chronic hepatitis B： An immunohistochemical study

AIM: To clarify the pathogenesis of ductular proliferation and its possible association with oval cell activation and hepatocyte regeneration. METHODS: Immunohistochemical staining and image analysis of the ductular structures in the liver tissues from 11 patients with severe chronic hepatitis B and 2 healthy individuals were performed. The liver specimens were sectioned serially, and then cytokeratin 8 (CK8), CK19, OV6, proliferating cell nuclear antigens (PCNA), glutathione-S-transferase (GST),α-fetal protein (AFP) and albumin were stained immunohistochemically. RESULTS: Typical and atypical types of ductular proliferation were observed in the portal tracts of the liver tissues in all 11 patients. The proliferating ductular cells were positive for CK8, CK19, OV6 and PCNA staining. Some atypical ductular cells displayed the morphological and immunohistochemical characteristics of hepatic oval cells. Some small hepatocyte-like cells were between hepatic oval cells and mature hepatocytes morphometri-cally and immunohistochemically. CONCLUSION: The proliferating ductules in the liver of patients with severe chronic liver disease may have different origins. Some atypical ductular cells are actually activated hepatic oval cells. Atypical ductular proliferation is related to hepatocyte regeneration and small hepatocyte-like cells may be intermediate transient cells between hepatic oval cells and mature hepatocytes.     

Antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo

Hepatic progenitor cells (called oval cells in rodents) proliferate during chronic liver injury. They have been suggested as targets of malignant transformation in chronic liver diseases, including chronic hepatitis C. Interferon alpha therapy reduces the risk of hepatocellular carcinoma (HCC) in chronic hepatitis C regardless of viral clearance. The aim of this study was to determine whether interferon alpha could reduce the risk of HCC by modifying preneoplastic events in the hepatic progenitor cell population. Pre- and post-treatment liver biopsies were evaluated for changes in t he hepaticprogenitor cell population in 16 patients with non-responding chronic hepatitis C Interferon alpha-based treatment significantly reduced the numbers of c-kit-positive hepatic progenitor cells by 50%. To determine the mechanism of cell number reduction, the effects of interferon alpha on murinehepatic progenitor cells were studied in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay and proliferating cell nuclear antigen staining showed that interferon alpha had a dose-dependent, anti-proliferative effect Interferon alpha stimulated hepatocytic and biliary differentiation of the oval cell lines reflected by increased expression of albumin and cytokeratin19 accompanied by decreased expression of alphafetoprotein and Thy-1. To validatethese results in vivo, mice were placed on the choline-deficient, ethionine-supplemented diet to induce liver injury and oval cell proliferation and treated with pegylated interferon alpha 2b for 2 weeks. This resulted in a significant four-fold reduction in the number of oval cells (P < .05). In conclusion, interferon alpha-based treatment reduced the number of hepatic progenitor cells in chronic liver injury by modulating apoptosis, proliferation, and differentiation. Supplementay material for this article can     

Isolation of fetal liver oval cells in physiologic conditions form their natural niche

The liver is characterized by a remarkable ability to proliferate and self-renew. In the situation of mild or moderate liver damage, hepatocytes carry out regeneration. Nevertheless, when liver damage is far too much extensive and the number of residual mature hepatocytes is not enough to accomplish regeneration, or likewise when mature hepatocyte proliferation is inhibited, hepatic regeneration depends on the activation of liver stem cells that give rise to oval cells. The population of liver stem cells is scant in normal liver. It is considered that in fetal liver this population is just over 1% of the cells. For this reason, it is necessary to isolate and enrich them for their study. With this goal several models of hepatic damage that permit the isolation of oval cells af ter the induction of massive hepatic injure have been developed. Here we present a simple methodology that allows the isolation of oval cells from rat fetal liver without prior induction of liver damage. The use of oval cell 2 (OC2) and oval cell 3 (OC3) antigens as molecular markers allowed the highly precise characterization of this cell population. Furthermore, the in vitro culture in presence of HGF yielded a substantial enrichment of the oval cell population.     

Inhibition of rat hepatocyte proliferation by transforming growth factor beta and glucagon is associated with inhibition of ERK2 and p70 S6 kinase

Stimulation of hepatocyte proliferation by epidermal growth factor (EGF) and insulin is inhibited by transforming growth factor beta (TGF-beta) and by glucagon. It is also suppressed by inhibitors of various protein kinases, including rapamycin, which blocks activation of p70 S6 kinase (p70(S6k)), PD98059, which inhibits the activation of extracellular-regulated kinase (ERK), and SB 203580, an inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated whether the inhibition of proliferation by TGF-beta involves these protein kinase cascades. Culture of hepatocytes with TGF-beta for 16 hours decreased the stimulation by EGF of ERK2 and p70(S6k) (by 50% and 35%, respectively), but did not affect the stimulation of either p38 MAPK, c-jun NH2-terminal kinase (JNK), or protein kinase B (PKB). Culture of hepatocytes with glucagon for 16 hours also inhibited the stimulation by EGF of activation of ERK2 and p70(S6k) (by approximately 50%). The inhibitory effects of glucagon were observed when the hormone was added either 10 minutes or 60 minutes before EGF addition, whereas no effects of TGF-beta were observed after 10-minute or 60-minute incubation. These results suggest that the inhibition of hepatocyte proliferation by TGF-beta may be in part mediated by inhibition of ERK2 and p70(S6k), but does not involve PKB, JNK, or p38 MAPK. Unlike glucagon, the effects of TGF-beta are not elicited in response to short-term treatment.     

Effect of intermediate-purity factor VIII (FVIII) concentrate on lymphocyte proliferation and apoptosis: transforming growth factor-beta is a significant immunomodulatory component of FVIII

Factor concentrates have been shown to have a variety of immunomodulatory effects in vitro. The presence of plasma-derived factor VIII (pdFVIII) has been shown to diminish lymphocyte proliferative response to mitogens. Recently, we have shown the presence of transforming growth factor-beta (TGF-beta) as an immunomodulatory component present in plasma-derived FVIII concentrate. However, the addition of neutralizing antibody to TGF-beta did not abrogate the inhibitory effect of pdFVIII on monocyte cytokine production, suggesting the presence of other, as yet undetermined, immunomodulatory agent/s in pdFVIII. To further characterize the immunomodulatory effects of pdFVIII, the in vitro effect of pdFVIII concentrate on proliferation and apoptosis of mitogen-stimulated T cells was studied using whole blood and purified T cells. The presence of pdFVIII increased the apoptosis of phytohaemagglutinn (PHA) -stimulated CD4 and CD8 T-cell subsets as determined by Annexin V binding and DNA fragmentation. T-cell subsets showed a pdFVIII dose-dependent inhibition of entry into S-phase and G(1) arrest. Addition of neutralizing anti-TGF-beta reduced some of these changes. To determine the physiological relevance of these findings, blood samples from five patients receiving FVIII prophylaxis were similarly studied ex vivo and showed significantly increased apoptosis of T-cell subsets as determined by Annexin V staining. TGF-beta has been reported to be a potent inhibitor of T-cell proliferation, arresting the cell cycle in G(1) phase and causing apoptosis. Together, these findings suggest that TGF-beta is a significant immunomodulatory component of pdFVIII concentrates.     

Oval cell proliferation in cirrhosis in rats. An experimental study

Aim: Oval cells are liver stem cells involved in liver regeneration following liver damage. Previous studies have shown that pretreatment with a hepatocyte inhibitor is required to allow full oval cell activation. This study investigates whether oval cells develop and proliferate in a model of experimental liver fibrosis without pretreatment with a known hepatocyte inhibitor. Methods: The study comprised 66 male Wistar rats divided into two groups: A (n = 6): controls; and B (n = 60): CCl(4) injection (intraperitoneally 2 mL/kg bodyweight 1:1 volume in corn oil twice weekly). Rats were sacrificed at four, eight and 12 weeks. Liver tissues were evaluated for the degree of fibrosis (Masson's trichrome), cell proliferation (Ki67 antigen), expression of alpha-fetoprotein (AFP) mRNA (RT-PCR and in situ hybridization), AFP protein (Western blot) and cytokeratin-19. Cells with morphologic features of oval cells that were cytokeratin 19 (CK19)+ and AFP mRNA+ were scored in morphometric analysis. Results: Oval cells were present in all 66 specimens; their percentage was higher in group B compared to group A (P < 0.001). AFP mRNA and protein expression increased as fibrosis advanced. Similarly, the numbers of CK19+, AFP mRNA+ and Ki67+ oval cells were higher in advanced fibrosis stages. Conclusion: This study demonstrates that oval cells develop and proliferate in a model of experimental liver fibrosis without pretreatment with a known hepatocytic inhibitor. However, further research is warranted in order to identify the exact molecular mechanisms involved in this process.     

Oval cell-mediated liver regeneration: Role of cytokines and growth factors

In experimental models, which induce liver damage and simultaneously block hepatocyte proliferation, the recruitment of a hepatic progenitor cell population comprised of oval cells is invariably observed. There is a substantial body of evidence to suggest that oval cells are involved in liver regeneration, as they differentiate into hepatocytes and biliary cells. Recently, bone marrow cells were shown to be a source of a stem cells with the capacity to repopulate the liver. Presently, the relationship between bone marrow cells and oval cells is unclear. Investigations will be greatly assisted by the availability of in vitro models based on a knowledge of cytokines that affect oval cells. While the cytokines, which regulate the different hematopoietic lineages, are well characterized, there is relatively little information regarding those that influence oval cells. This review outlines recent developments in the field of oval cell research and focuses on cytokines and growth factors that have been implicated in regulating oval cell proliferation and differentiation.     

Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration.

Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation.     

Extracellular ATP activates c-jun N-terminal kinase signaling and cell cycle progression in hepatocytes

Partial hepatectomy leads to an orchestrated regenerative response, activating a cascade of cell signaling events necessary for cell cycle progression and proliferation of hepatocytes. However, the identity of the humoral factors that trigger the activation of these pathways in the concerted regenerative response in hepatocytes remains elusive. In recent years, extracellular ATP has emerged as a rapidly acting signaling molecule that influences a variety of liver functions, but its role in hepatocyte growth and regeneration is unknown. In this study, we sought to determine if purinergic signaling can lead to the activation of c-jun N-terminal kinase (JNK), a known central player in hepatocyte proliferation and liver regeneration. Hepatocyte treatment with ATPgammaS, a nonhydrolyzable ATP analog, recapitulated early signaling events associated with liver regeneration-that is, rapid and transient activation of JNK signaling, induction of immediate early genes c-fos and c-jun, and activator protein-1 (AP-1) DNA-binding activity. The rank order of agonist preference, UTP>ATP>ATPgammaS, suggests that the effects of extracellular ATP is mediated through the activation of P2Y2 receptors in hepatocytes. ATPgammaS treatment alone and in combination with epidermal growth factor (EGF) substantially increased cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expression and hepatocyte proliferation in vitro. Extracellular ATP as low as 10 nM was sufficient to potentiate EGF-induced cyclin D1 expression. Infusion of ATP by way of the portal vein directly activated hepatic JNK signaling, while infusion of a P2 purinergic receptor antagonist prior to partial hepatectomy inhibited JNK activation. In conclusion, extracellular ATP is a hepatic mitogen that can activate JNK signaling and hepatocyte proliferation in vitro and initiate JNK signaling in regenerating liver in vivo. These findings have implications for enhancing our understanding of novel factors involved in the initiation of regeneration, liver growth, and development.     

Endothelial cells provide feedback control for vascular remodeling through a mechanosensitive autocrine TGF-beta signaling pathway

Mechanical forces are potent modulators of the growth and hypertrophy of vascular cells. We examined the molecular mechanisms through which mechanical force and hypertension modulate endothelial cell regulation of vascular homeostasis. Exposure to mechanical strain increased the paracrine inhibition of vascular smooth muscle cells (VSMCs) by endothelial cells. Mechanical strain stimulated the production of perlecan and heparan sulfate glycosaminoglycans by endothelial cells. By inhibiting the expression of perlecan with an antisense vector we demonstrated that perlecan was essential to the strain-mediated effects on endothelial cell growth control. Mechanical regulation of perlecan expression in endothelial cells was governed by a mechanotransduction pathway requiring autocrine transforming growth factor beta (TGF-beta) signaling and intracellular signaling through the ERK pathway. Immunohistochemical staining of the aortae of spontaneously hypertensive rats demonstrated strong correlations between endothelial TGF-beta, phosphorylated signaling intermediates, and arterial thickening. Further, studies on ex vivo arteries exposed to varying levels of pressure demonstrated that ERK and TGF-beta signaling were required for pressure-induced upregulation of endothelial HSPG. Our findings suggest a novel feedback control mechanism in which net arterial remodeling to hemodynamic forces is controlled by a dynamic interplay between growth stimulatory signals from VSMCs and growth inhibitory signals from endothelial cells.     

Further studies on the biological activities of the CFU-S inhibitory tetrapeptide AcSDKP. II. Unresponsiveness of isolated adult rat hepatocytes, 3T3, FDC-P2, and K562 cell lines to AcSDKP. Possible involvement of intermediary cell(s) in the mechanism of AcSDKP action

Because the molecular mechanisms of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP; an inhibitor of spleen colony-forming unit [CFU-S] DNA synthesis) are difficult to study on bone marrow due to the scarcity of CFU-S in this tissue, we sought a pure cell population responsive to the molecule in vitro. Although growth factor-stimulated DNA synthesis in primary culture of hepatocytes and Balb/c 3T3 cells can be inhibited by transforming growth factor beta (TGF beta) and interferon alpha/beta (IFN[alpha/beta], respectively, neither hepatocytes nor 3T3 cells were found to be sensitive to AcSDKP. DNA synthesis in stimulated murine FDC-P2 cell lines and in human K562 cell lines also remained unchanged after exposure to the tetrapeptide. The fact that hepatocytes do respond in vivo to AcSDKP implies the existence of intermediary cell(s) involved in AcSDKP action in vivo that are lacking in hepatocyte culture. Whether intermediary cell(s) are implicated in the inhibitory action of AcSDKP on CFU-S entry into DNA synthesis is now being investigated.