Hepatic Vascular Endothelial Growth Factor Research Articles

Cinnamaldehyde Relieves Induced Hepatocellular Carcinoma in Rat Model via Targeting Wnt/β-Catenin Pathway

Cinnamaldehyde (CA) is a natural compound that has promising biological activity. The current study investigates the antitumor activity of CA in thioacetamide induced hepatocellular carcinoma (HCC) in rats through targeting the Wnt/β-catenin pathway and evaluates the capability of CA to relieve hepatocytes oxidative stress in the HCC-rat model. After 16 weeks of HCC induction by thioacetamide (TAA), rats were treated for 7 consecutive weeks with CA daily; i.p. injection, Alpha-fetoprotein (AFP) level, necroinflammatory score and fibrosis percentage were measured to assess HCC development. The Wnt/β-catenin pathway was evaluated by measuring the hepatic protein level of Wnt-3a, β-catenin, cyclin D, matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF). Furthermore, hepatocytes’ oxidative stress was assessed by measuring hepatic GSH and MDA contents. Results showed that CA was significantly inhibiting the Wnt/β-catenin pathway through the downregulation of hepatic Wnt-3a, β-catenin, cyclin D, MMP-9, and VEGF. Moreover, CA ameliorates hepatocytes’ oxidative stress via lowering hepatic MDA content and rising hepatic GSH content. Thus, in conclusion, CA is a promising treatment for HCC. It not only has an effective antitumor activity but also ameliorates hepatocytes’ oxidative stress.

Susceptibility of Rat Steatotic Liver to Ischemia-Reperfusion Is Treatable With Liver-Selective Matrix Metalloproteinase Inhibition.

This study examined whether enhanced susceptibility of steatotic liver to ischemia-reperfusion (I/R) injury is due to impaired recruitment of bone marrow (BM) progenitors of liver sinusoidal endothelial cells (LSECs, also called sinusoidal endothelial cell progenitor cells [sprocs]) with diminished repair of injured LSECs and whether restoring signaling to recruit BM sprocs reduces I/R injury. Hepatic vessels were clamped for 1hour in rats fed a high-fat, high-fructose (HFHF) diet for 5, 10, or 15weeks. Matrix metalloproteinase 9 (MMP-9) antisense oligonucleotides (ASO) or an MMP inhibitor were used to induce liver-selective MMP-9 inhibition. HFHF rats had mild, moderate, and severe steatosis, respectively, at 5, 10, and 15weeks. I/R injury was enhanced in HFHF rats; this was accompanied by complete absence of hepatic vascular endothelial growth factor (VEGF)-stromal cell-derived factor 1 (sdf1) signaling, leading to lack of BM sproc recruitment. Liver-selective MMP-9 inhibition to protect against proteolytic cleavage of hepatic VEGF using either MMP-9 ASO or intraportal MMP inhibitor in 5-week and 10-week HFHF rats enhanced hepatic VEGF-sdf1 signaling, increased BM sproc recruitment, and reduced alanine aminotransferase (ALT) by 92% and 77% at 5weeks and by 80% and 64% at 10weeks of the HFHF diet, respectively. After I/R injury in 15-week HFHF rats, the MMP inhibitor reduced active MMP-9 expression by 97%, ameliorated histologic evidence of injury, and reduced ALT by 58%, which is comparable to control rats sustaining I/R injury. Rescue therapy with intraportal MMP inhibitor, given after ischemia, in the 5-week HFHF rat reduced ALT by 71% and reduced necrosis. Lack of signaling to recruit BM sprocs that repair injured LSECs renders steatotic liver more susceptible to I/R injury. Liver-selective MMP-9 inhibition enhances VEGF-sdf1 signaling and recruitment of BM sprocs, which markedly protects against I/R injury, even in severely steatotic rats.

Expression of VEGF and Nestin in Chronic Restraint-Induced Stress in the Livers of Male Albino Rats.

Background and Aim of work: Stress and its related comorbid diseases are responsible for many liver pathologies. Nestin, a protein detected in a wide range of progenitor cells, was implicated in hepatic remodeling. Moreover, vascular endothelial growth factor (VEGF) is involved in vessel repair. The aim of this study was to evaluate the effect of chronic restrain stress (CRS) on expression of hepatic VEGF and Nestin. Materials and methods: Twelve male rats were divided into two groups; negative control and CRS. Animals of CRS were restrained individually in ventilated glass tube restrainers daily for 6 hours/day for 3 weeks. The liver samples were stained by hematoxylin and eosin, Masson trichrome and immunohistochemical staining for Nestin and VEGF with morphometric analysis. Results: Exposure to chronic restraint stress for 3 weeks caused hepatic inflammation, deposition of collagen. Immunohistochemical staining showed a coinciding significantly higher positive reaction for VEGF and Nestin in the CRS group. Conclusion: Vascular endothelial growth factor and Nestin immunohistochemical staining were both increased in chronic restraint-induced hepatic stress coinciding with the induced increase in hepatic inflammation, deposition of collagen. Nestin & VEGF

Downregulation of flt-1 and HIF-1α Gene Expression by Some Antioxidants in Rats Under Sodium Nitrite-Induced Hypoxic Stress.

This study assessed the effect of L-arginine (L-argin), carnosine (carno), or their combination in the amelioration of certain biochemical indices induced in the liver of hypoxic rats. Hypoxia was induced via sodium nitrite (S.nit) injection at a dose of 75 mg/kg. Rats were administered L-argin (250 mg/kg) or carno (250 mg/kg), either alone or in combination, 24 hours and 1 hour prior to S.nit intoxication. Hypoxia significantly elevated serum alanine aminotransferase, in addition to a significant upregulation of hepatic heat shock protein 70 with concurrent reduction in the level of vascular endothelial growth factor. Moreover, hepatic vascular endothelial growth factor 1 (flt-1), hypoxia inducible factor-1α gene expression, and cytochrome P450 levels were elevated, compared with the normoxic group. The antioxidants, administered either alone or in combination, markedly downregulated all of the previously mentioned biomarkers, compared to the hypoxic rats. Histopathological examination revealed hepatocellular degeneration and nuclear pyknosis, in addition to inflammatory cellular infiltration in the hypoxic rats, whereas treatment with the studied antioxidants improved the liver architecture. The present data revealed the efficacy of L-argin and carno in ameliorating the hepatic damage induced via angiogenic markers in response to hypoxia, the combination regimen showing the superior effect.

Expression of VEGF and Nestin in Chronic Restraint-Induced Stress in the Livers of Male Albino Rats.

Background and Aim of work: Stress and its related comorbid diseases are responsible for many liver pathologies. Nestin, a protein detected in a wide range of progenitor cells, was implicated in hepatic remodeling. Moreover, vascular endothelial growth factor (VEGF) is involved in vessel repair. The aim of this study was to evaluate the effect of chronic restrain stress (CRS) on expression of hepatic VEGF and Nestin. Materials and methods: Twelve male rats were divided into two groups; negative control and CRS. Animals of CRS were restrained individually in ventilated glass tube restrainers daily for 6 hours/day for 3 weeks. The liver samples were stained by hematoxylin and eosin, Masson trichrome and immunohistochemical staining for Nestin and VEGF with morphometric analysis. Results: Exposure to chronic restraint stress for 3 weeks caused hepatic inflammation, deposition of collagen. Immunohistochemical staining showed a coinciding significantly higher positive reaction for VEGF and Nestin in the CRS group. Conclusion: Vascular endothelial growth factor and Nestin immunohistochemical staining were both increased in chronic restraint-induced hepatic stress coinciding with the induced increase in hepatic inflammation, deposition of collagen. Nestin & VEGF

Antiangiogenic activity of vitexicarpine in experimentally induced hepatocellular carcinoma: Impact on vascular endothelial growth factor pathway.

Angiogenesis plays important roles in progression of hepatocellular carcinoma. The antiangiogenic mechanisms of vitexicarpine are not fully defined. Therefore, we conducted the following study to evaluate the antiangiogenic mechanism and antitumor activity of vitexicarpine in vivo model of hepatocellular carcinoma through modulation of vascular endothelial growth factor signaling pathway. Hepatocellular carcinoma was induced in Sprague Dawley rats by thioacetamide. Hepatocellular carcinoma was assessed by measuring serum alpha-fetoprotein and investigating liver sections stained with hematoxylin/eosin. Hepatocellular carcinoma rats were injected with vitexicarpine (150 mg/kg) for 2 weeks. Hepatic vascular endothelial growth factor was measured by enzyme-linked immunosorbent assay. Protein and expression of hepatic phospho-Ser473-AKT (p-AKT) and phospho-Tyr419-Src (p-Src) were determined. The apoptotic pathway was evaluated by assessment of protein expression of caspase-3. Vitexicarpine increased rats' survival time and decreased serum alpha-fetoprotein as well as it ameliorated fibrosis and massive hepatic tissue breakdown. It attenuated hepatocellular carcinoma-induced protein and gene expression of vascular endothelial growth factor, p-AKT, p-Src, and caspase-3. In conclusion, this study suggests that vitexicarpine possesses both antiangiogenic and antitumor activities through inhibition of vascular endothelial growth factor, p-AKT/AKT, and p-Src with subsequent inhibition of apoptotic pathway.

Acute hypoxia up-regulates HIF-1α and VEGF mRNA levels in Amazon hypoxia-tolerant Oscar (Astronotus ocellatus).

Amazon fish maintain oxygen uptake through a variety of strategies considered evolutionary and adaptive responses to the low water oxygen saturation, commonly found in Amazon waters. Oscar (Astronotus ocellatus) is among the most hypoxia-tolerant fish in Amazon, considering its intriguing anaerobic capacity and ability to depress oxidative metabolism. Previous studies in hypoxia-tolerant and non-tolerant fish have shown that hypoxia-inducible factor-1α (HIF-1α) gene expression is positively regulated during low oxygen exposure, affecting vascular endothelial growth factor (VEGF) transcription and fish development or tolerance in different manners. However, whether similar isoforms exists in tolerant Amazon fish and whether they are affected similarly to others physiological responses to improve hypoxia tolerance remain unknown. Here we evaluate the hepatic HIF-1α and VEGF mRNA levels after 3h of acute hypoxia exposure (0.5mgO2/l) and 3h of post-hypoxia recovery. Additionally, hematological parameters and oxidative enzyme activities of citrate synthase (CS) and malate dehydrogenase (MDH) were analyzed in muscle and liver tissues. Overall, three sets of responses were detected: (1) as expected, hematocrit, hemoglobin concentration, red blood cells, and blood glucose increased, improving oxygen carrying capacity and glycolysis potential; (2) oxidative enzymes from liver decreased, corroborating the tendency to a widespread metabolic suppression; and (3) HIF-1α and VEGF increased mRNA levels in liver, revealing their role in the oxygen homeostasis through, respectively, activation of target genes and vascularization. This is the first study to investigate a hypoxia-related transcription factor in a representative Amazon hypoxia-tolerant fish and suggests that HIF-1α and VEGF mRNA regulation have an important role in enhancing hypoxia tolerance in extreme tolerant species.

VEGF-sdf1 recruitment of CXCR7+ bone marrow progenitors of liver sinusoidal endothelial cells promotes rat liver regeneration.

In liver injury, recruitment of bone marrow (BM) progenitors of liver sinusoidal endothelial cells (sprocs) is necessary for normal liver regeneration. Hepatic vascular endothelial growth factor (VEGF) is a central regulator of the recruitment process. We examine whether stromal cell-derived factor 1 [sdf1, or CXC ligand 12 (CXCL12)] acts downstream from VEGF to mediate recruitment of BM sprocs, what the sdf1 receptor type [CXC receptor (CXCR)-4 or CXCR7] is on sprocs, and whether sdf1 signaling is required for normal liver regeneration. Studies were performed in the rat partial hepatectomy model. Tracking studies of BM sprocs were performed in wild-type Lewis rats that had undergone BM transplantation from transgenic enhanced green fluorescent protein-positive Lewis rats. Knockdown studies were performed using antisense oligonucleotides (ASOs). Expression of sdf1 doubles in liver and liver sinusoidal endothelial cells (LSECs) after partial hepatectomy. Upregulation of sdf1 expression increases proliferation of sprocs in the BM, mobilization of CXCR7(+) BM sprocs to the circulation, and engraftment of CXCR7(+) BM sprocs in the liver and promotes liver regeneration. Knockdown of hepatic VEGF with ASOs decreases hepatic sdf1 expression and plasma sdf1 levels. When the effect of VEGF knockdown on sdf1 is offset by infusion of sdf1, VEGF knockdown-induced impairment of BM sproc recruitment after partial hepatectomy is completely attenuated and liver regeneration is normalized. These data demonstrate that the VEGF-sdf1 pathway regulates recruitment of CXCR7(+) BM sprocs to the hepatic sinusoid after partial hepatectomy and is required for normal liver regeneration.

The effects of Tao-Hong-Si-Wu on hepatic necroinflammatory activity and fibrosis in a murine model of chronic liver disease

BackgroundTao-Hong-Si-Wu decoction (THSWD) is a traditional Chinese herbal medicine that has been used for centuries in the treatment of Chinese patients with chronic liver disease. Recently, THSWD has been reported to alter vascular endothelial growth factor (VEGF) induced angiogenesis, raising the possibility that in addition to its anti-inflammatory properties; THSWD might also inhibit hepatic blood flow associated fibrosis. AimTo document the effects of THSWD on hepatic necroinflammatory disease activity, fibrosis and VEGF signaling in a murine model of chronic liver disease. MethodsSixty adult mice were equally divided into six study groups. Five groups were exposed to subcutaneous carbon tetrachloride (0.1ml/10g BW) for six weeks. Three of the five groups were treated with different concentrations of THSWD (4.25, 8.50, 17.00g/kg), one with 0.1mg/kg of Colchicine (positive control), and one with physiologic saline (negative control). Mice in the sixth group were not exposed to CCl4 and remained untreated (healthy controls). Liver enzymes/function tests, hyaluronic acid and laminin levels were measured in serum, and hepatic histology, VEGF, Flt-1 and kinase insert domain-containing receptor (KDR), Akt and phosphorylated Akt (pAkt) expression were documented in liver tissue at the end of treatment. ResultsHepatic necroinflammatory disease activity and fibrosis were significantly attenuated in THSWD treated mice in a dose dependent manner. These beneficial results were similar and often exceeded those achieved with Colchicine. In addition, VEGF, Flt-1, KDR, Akt and pAkt mRNA and protein expression were reduced in TSHWD treated mice. ConclusionsIn this animal model of chronic liver disease, THSWD decreased hepatic necroinflammatory disease and fibrosis. Inhibition of VEGF expression and downstream signaling were associated with these findings. Further studies with this and other TCMs as treatment for chronic liver disease are warranted.

The Effects of Taoren-Honghua Herb Pair on Pathological Microvessel and Angiogenesis-Associated Signaling Pathway in Mice Model of CCl4-Induced Chronic Liver Disease.

Chronic liver disease is one of the most common diseases that threaten human health. Effective treatment is still lacking in western medicine. Semen Persicae (Taoren) and Flos Carthami (Honghua) are known to relieve acute hepatic injury and inflammation, improve microcirculation, and reduce tissue fiber. The aim of our study is to investigate the potential mechanisms of Taoren-Honghua Herb Pair (THHP) in murine model of chronic liver disease caused by Carbon Tetrachloride (CCl4). Mice were randomly divided into seven groups: (1) blank, (2) model, (3) control (colchicine, 0.1 mg/kg), (4) THHP (5.53, 2.67, and 1.33 g/kg), and (5) Tao Hong Siwu Decoction (THSWD) (8.50 g/kg). Histological change and microvessels density were examined by microscopy. Hepatic function, serum fibrosis related factors, and hepatic vascular endothelial growth factor (VEGF) were measured with ELISA. VEGF, kinase insert domain-containing receptor (KDR), Flt-1, and Akt mRNA expression in hepatic tissue were determined with PCR. Tissues of Akt, pAkt, KDR, and Flt-1 were measured with western blotting. Data from this study showed that THHP improved hepatic function and restrained the hepatic inflammation and fibrosis. Its role in inhibiting pathological angiogenesis and hepatic fibrogenesis may be through affecting the angiogenesis-associated VEGF and its upstream and downstream signaling pathways.

Cilostazol improves hepatic blood perfusion, microcirculation, and liver regeneration after major hepatectomy in rats.

Major hepatectomy or small-for-size liver transplantation may result in postoperative liver failure. So far, no treatment is available to improve liver regeneration. Herein, we studied whether cilostazol, a selective phosphodiesterase III inhibitor, is capable of improving liver regeneration after major hepatectomy. Sprague-Dawley rats (n = 74) were treated with cilostazol (5 mg/kg daily) or a glucose solution and underwent either 70% liver resection or a sham operation. Before and after surgery, hepatic arterial and portal venous blood flow and hepatic microvascular perfusion were analyzed. Liver morphology, function, and regeneration were studied with histology, immunohistochemistry, western blotting, and bile excretion analysis. Cilostazol significantly increased hepatic blood flow and microcirculation before and after hepatectomy in comparison with sham-operated controls. This was associated with an elevation of hepatic vascular endothelial growth factor expression, an increase of hepatocellular proliferation, and an acceleration of liver regeneration. Furthermore, cilostazol protected the tissue of the remnant liver as indicated by an attenuation of hepatocellular disintegration. In conclusion, cilostazol increases hepatic blood perfusion, microcirculation, and liver regeneration after a major hepatectomy. Thus, cilostazol may represent a novel strategy to reduce the rate of liver failure after both extended hepatectomy and small-for-size liver transplantation.

Resolution of liver fibrosis requires myeloid cell-driven sinusoidal angiogenesis.

Angiogenesis is a key feature of liver fibrosis. Although sinusoidal remodeling is believed to contribute to fibrogenesis, the impact of sinusoidal angiogenesis on the resolution of liver fibrosis remains undefined. Myeloid cells, particularly macrophages, constantly infiltrate the fibrotic liver and can profoundly contribute to remodeling of liver sinusoids. We observe that the development of fibrosis is associated with decreased hepatic vascular endothelial growth factor (VEGF) expression as well as sinusoidal rarefication of the fibrotic scar. In contrast, the resolution of fibrosis is characterized by a rise in hepatic VEGF levels and revascularization of the fibrotic tissue. Genetic ablation of VEGF in myeloid cells or pharmacological inhibition of VEGF receptor 2 signaling prevents this angiogenic response and the resolution of liver fibrosis. We observe increased expression of matrix metalloproteases as well as decreased expression of tissue inhibitor of metalloproteases confined to sinusoidal endothelial cells in response to myeloid cell VEGF. Remarkably, reintroduction of myeloid cell-derived VEGF upon recovery restores collagenolytic acitivity and the resolution of fibrosis. We identify myeloid cell-derived VEGF as a critical regulator of extracellular matrix degradation by liver endothelial cells, thereby unmasking an unanticipated link between angiogenesis and the resolution of fibrosis.

Markers of angiogenesis associated with surgical attenuation of congenital portosystemic shunts in dogs.

BackgroundDogs with congenital portosystemic shunts (CPSS) have hypoplasia of the intrahepatic portal veins. Surgical CPSS attenuation results in the development of the intrahepatic portal vasculature, the precise mechanism for which is unknown, although new vessel formation by angiogenesis is suspected.HypothesisThat the degree of portal vascular development and the increase in portal vascularization after CPSS attenuation is significantly associated with hepatic vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) gene expression and serum VEGF concentration.AnimalsClient‐owned dogs with CPSS undergoing surgical treatment. Forty‐nine dogs were included in the gene expression data and 35 in the serum VEGF data.Materials and MethodsDogs surgically treated by partial or complete CPSS attenuation were prospectively recruited. Relative gene expression of VEGF and VEGFR2 was measured in liver biopsy samples taken at initial and follow‐up surgery using quantitative polymerase chain reaction. Serum VEGF concentration was measured before and after CPSS attenuation using a canine specific ELISA. Statistical significance was set at the 5% level (P ≤ .05).ResultsThere was a significant increase in the mRNA expression of VEGFR2 after partial attenuation (P = .006). Dogs that could tolerate complete attenuation had significantly greater VEGFR2 mRNA expression than those that only tolerated partial attenuation (P = .037). Serum VEGF concentration was significantly increased at 24 (P < .001) and 48 (P = .003) hours after attenuation.Conclusions and Clinical ImportanceThese findings suggest that intrahepatic angiogenesis is likely to occur after the surgical attenuation of CPSS in dogs, and contributes to the development of the intrahepatic vasculature postoperatively.

Preventive effects of dexmedetomidine on the liver in a rat model of acid-induced acute lung injury.

The aim of this study was to examine whether dexmedetomidine improves acute liver injury in a rat model. Twenty-eight male Wistar albino rats weighing 300–350 g were allocated randomly to four groups. In group 1, normal saline (NS) was injected into the lungs and rats were allowed to breathe spontaneously. In group 2, rats received standard ventilation (SV) in addition to NS. In group 3, hydrochloric acid was injected into the lungs and rats received SV. In group 4, rats received SV and 100 µg/kg intraperitoneal dexmedetomidine before intratracheal HCl instillation. Blood samples and liver tissue specimens were examined by biochemical, histopathological, and immunohistochemical methods. Acute lung injury (ALI) was found to be associated with increased malondialdehyde (MDA), total oxidant activity (TOA), oxidative stress index (OSI), and decreased total antioxidant capacity (TAC). Significantly decreased MDA, TOA, and OSI levels and significantly increased TAC levels were found with dexmedetomidine injection in group 4 (P < 0.05). The highest histologic injury scores were detected in group 3. Enhanced hepatic vascular endothelial growth factor (VEGF) expression and reduced CD68 expression were found in dexmedetomidine group compared with the group 3. In conclusion, the presented data provide the first evidence that dexmedetomidine has a protective effect on experimental liver injury induced by ALI.

Targeting different angiogenic pathways with combination of curcumin, leflunomide and perindopril inhibits diethylnitrosamine-induced hepatocellular carcinoma in mice

No effective chemopreventive agent has been approved against hepatocellular carcinoma (HCC) to date. Since HCC is one of the hypervascular solid tumors, blocking angiogenesis represents an intriguing approach to HCC chemoprevention. The aim of the current study was to examine the combined effect of the anti-angiogenic agents: leflunomide; a disease modifying antirheumatic drug, perindopril; an angiotensin converting enzyme inhibitor (ACEI) and curcumin; the active principle of turmeric, on diethylnitrosamine (DEN)-induced HCC in mice. Eight weeks following DEN administration, there was a significant rise in immunohistochemical staining of CD31-positive endothelial cells and consequently hepatic microvessel density (MVD) as compared to normal liver. DEN treatment was associated with elevation in hepatic vascular endothelial growth factor (VEGF) level as compared to normal controls (P<0.05, 3842±72pg/ml and 2520.8±97pg/ml, respectively). Similarly, increased hepatic expression of hypoxia inducible growth factor-1α (HIF-1α) was observed in 100% of the DEN-treated animals compared to 0% in their normal counterparts. Treatment with leflunomide, perindopril or curcumin alone abrogated the DEN-induced increased MVD as well as the elevated expression of VEGF, while only curcumin inhibited HIF-1α hepatic expression. Combination of these agents showed further inhibitory action on neovascularization and synergistic attenuation of hepatic VEGF (1954.27±115pg/ml) when compared to each single agent. Histopathological examination revealed a more beneficial chemopreventive activity in the combination group compared to each monotherapy. In conclusion, the combination treatment of leflunomide, perindopril and curcumin targeting different angiogenic pathways, resulted in synergistic inhibition of angiogenesis and consequently more effective chemoprevention of HCC.

Hepatic Vascular Endothelial Growth Factor Regulates Recruitment of Rat Liver Sinusoidal Endothelial Cell Progenitor Cells

After liver injury, bone marrow-derived liver sinusoidal endothelial cell progenitor cells (BM SPCs) repopulate the sinusoid as liver sinusoidal endothelial cells (LSECs). After partial hepatectomy, BM SPCs provide hepatocyte growth factor, promote hepatocyte proliferation, and are necessary for normal liver regeneration. We examined how hepatic vascular endothelial growth factor (VEGF) regulates recruitment of BM SPCs and their effects on liver injury. Rats were given injections of dimethylnitrosamine to induce liver injury, which was assessed by histology and transaminase assays. Recruitment of SPCs was analyzed by examining BM SPC proliferation, mobilization to the circulation, engraftment in liver, and development of fenestration (differentiation). Dimethylnitrosamine caused extensive denudation of LSECs at 24 hours, followed by centrilobular hemorrhagic necrosis at 48 hours. Proliferation of BM SPCs, the number of SPCs in the bone marrow, and mobilization of BM SPCs to the circulation increased 2- to 4-fold by 24 hours after injection of dimethylnitrosamine; within 5 days, 40% of all LSECs came from engrafted BM SPCs. Allogeneic resident SPCs, infused 24 hours after injection of dimethylnitrosamine, repopulated the sinusoid as LSECs and reduced liver injury. Expression of hepatic VEGF messenger RNA and protein increased 5-fold by 24 hours after dimethylnitrosamine injection. Knockdown of hepatic VEGF with antisense oligonucleotides completely prevented dimethylnitrosamine-induced proliferation of BM SPCs and their mobilization to the circulation, reduced their engraftment by 46%, completely prevented formation of fenestration after engraftment as LSECs, and exacerbated dimethylnitrosamine injury. BM SPC recruitment is a repair response to dimethylnitrosamine liver injury in rats. Hepatic VEGF regulates recruitment of BM SPCs to liver and reduces this form of liver injury.

Pregabalin Induces Hepatic Hypoxia and Increases EndothelialCell Proliferation in Mice, a Process Inhibited by DietaryVitamin E Supplementation

The preceding article identified key components of pregabalin's mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.

Echinomycin Decreases Induction of Vascular Endothelial Growth Factor and Hepatocyte Regeneration in Acetaminophen Toxicity in Mice

Up-regulation of vascular endothelial growth factor (VEGF) is important to hepatocyte regeneration in the late stages of acetaminophen (APAP) toxicity in the mouse. This study was conducted to examine the relationship of hypoxia-inducible factor 1α (HIF-1α) to VEGF and hepatocyte regeneration in APAP toxicity using an inhibitor of HIF-1α DNA-binding activity, echinomycin (EC). B6C3F1 male mice were treated with APAP (200 mg/kg IP), followed by EC (0.15 mg IP) and killed at 4 hr. Serum alanine aminotransferase (ALT), necrosis, hepatic glutathione (GSH) and APAP protein adducts were comparable in the APAP/EC and the APAP/veh mice at 4 hr. Additional studies showed that high dose EC (0.3 mg) reduced hepatic VEGF but also lowered hepatic GSH. Subsequent studies were performed using the 0.15-mg dose of EC. Although EC 0.15 mg had no effect on hepatic VEGF levels at 8 hr, by 24 hr VEGF levels were decreased by 40%. Toxicity (ALT and histopathology) was comparable in the APAP and APAP/EC groups at 24 and 48 hr. Proliferating cell nuclear antigen expression was reduced by both Western blot analysis and immunohistochemical staining in the APAP/EC mice at 48 hr. The data support the hypothesis that induction of HIF-1α, its binding to DNA and subsequent expression of VEGF are important factors in hepatocyte regeneration in APAP toxicity in the mouse.

Abstract 2470: Clinically used selective aldosterone blocker attenuates hepatocarcinogenesis via angiogenesis inhibition in rats

Abstract Hepatocellular carcinoma (HCC) is now a major health problem. Since the high-risk group of either primary or secondary HCC development seems to be clearer than in the other types of tumors, it is likely that chemopreventive agents will be beneficial in improving the prognosis of HCC. In addition to assessment of tumor growth, recent studies have demonstrated that angiogenesis plays a pivotal role in the hepatocarcinogenesis process. Although several anti-angiogenic agents have already been employed in the clinical practice, most patients show adverse reactions with new drugs. An alternative approach may be to find a clinically available compound that also exhibits anti-angiogenic activity for which the safety of long-term administration has been proven. We previously reported that angiotensin-II (AT-II) was a prerequisite for hepatocarcinogenesis. Suppression of AT-II by angiotensin-converting enzyme inhibitor attenuates hepatocarcinogenesis via-suppression of neovascularization in the clinical practice. Recent studies have also suggested possible involvement of aldosterone (Ald) in neovascularization. The aim of our current study was to elucidate the effect of the clinically used eplerenone, a selective Ald blocker (SAB), on the rat hepatocarcinogenesis especially in conjunction with angiogenesis. We employed the rat hepatocarcinogenesis model, induced by feeding the rats on a choline-deficient, L-amino acid-defined diet without any carcinogen (CDAA model). The effects of SAB on the development of liver enzyme (namely, the placental form of glutathione-S-transferase (GST-P))-altered pre-neoplastic lesions and angiogenesis were examined. We also performed an in-vitro study to elucidate the possible mechanisms involved. Treatment with SAB markedly inhibited GST-P-positive lesions along with suppression of neovascularization. The hepatic vascular endothelial growth factor (VEGF) was also significantly attenuated. Our in-vitro study showed that Ald augmented both proliferation and tubule formation of the endothelial cells, and that SAB inhibited both activities in a dose-dependent manner via inhibition of phosphorylation of the extracellular signal-regulated kinase 1/2. These results indicated that, as well as AT-II, Ald plays a pivotal role in hepatocarcinogenesis, and that augmentation of angiogenesis is involved. Since SAB is already widely used in the clinical practice with proved safety, this drug may represent a potential new strategy for chemoprevention against HCC in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2470.

Angioprevention is Implicated in Resveratrol Chemoprevention of Experimental Hepatocarcinogenesis

Hepatocellular carcinoma (HCC), a hypervascular tumor, is one of the most common and lethal cancers worldwide. We previously showed that resveratrol, a dietary polyphenol, inhibits rat liver carcinogenesis through antioxidative and antiinfl ammatory mechanisms. As resveratrol possesses antiangiogenic properties, we hypothesize that it may exert chemoprevention of hepatocarcinogenesis by suppressing angiogenesis. The antiangiogenic effect of resveratrol was investigated by analyzing livers from our previous study in which resveratrol (50-300 mg/kg) exerted chemopreventive action against diethylnitrosamine (DENA)-induced rat liver tumorigenesis. Hepatic angiogenesis was evaluated by microvessel density (MVD) based on immunohistochemical staining of CD31-positive endothelial cells. The expression of hepatic vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by immunohistochemistry. Sixteen weeks following the administration of DENA, there was a substantial increase in hepatic MVD as compared to normal liver. A dramatic increase in hepatic VEGF and HIF-1α was observed in DENA-treated animals compared to normal counterparts. Treatment with resveratrol dose-dependently abrogated the DENA-induced increased MVD as well as the elevated expression of VEGF and HIF-1α. DENA-initiated hepatocarcinogenesis in rats exhibits substantial neovascularization possibly due to overexpression of VEGF upregulated by HIF-1α. Resveratrol exerts a remarkable angiosuppressive effect in DENA-evoked hepatocellular carcinogenesis. Resveratrol-mediated inhibition of angiogenesis could be achieved by suppressing VEGF expression through downregulation of HIF1α. These results, in conjunction with our previous fi ndings, provide evidence that angiosuppression is involved in resveratrol-mediated chemoprevention of rat liver carcinogenesis and support the potential use of this natural agent in the prevention and therapy of HCC.