Antiangiogenic activity of curcumin in hepatocellular carcinoma cells implanted nude mice.

Antiangiogenic activity of curcumin on the tumor neogenesis was investigated by evaluating the density of neocapillaries induced by Hepatocellular carcinoma cells (HepG2) in mice, using intravital fluorescence videomicroscopy. Male BALB/c nude mice (20-25 g) were used, and a dorsal skin-fold chamber was implanted. HepG2 (30 microl of 2 x 10(6) cells) were inoculated on the upper surface of the skin within the chamber. The mice were divided into two groups as follows. Dimethyl sulfoxide solution (0.1%) was fed (HepG2 group, n=5) or curcumin solution (3000 mg/kg bw) was fed oral daily (HepG2-Cur group, n=5), one day after the inoculation of HepG. On days 7 and 14 post-tumor-inoculation, the tumor microvasculature was visualized by injecting 0.1 ml of 0.5% rhodamine B isothiocyanate-labeled dextran intravenously, and observed under an intravital fluorescence videomicroscope. Based on the recorded videoimage, the tumor neocapillary density and microvasculature were evaluated using a digital image analysis and correlated with the tumor area. The image analysis demonstrated that in the HepG2-group the neocapillary densities were significantly increased on day 7, and day 14, compared to the aged-matched Sham-group (P<0.05). In the HepG2-Cur group, the increase of tumor neocapillary density was attenuated significantly. It was suggested that high dose of curcumin might be an effective anti-angiogenic drug in the treatment against tumor.

Tumor neocapillary density in hepatocellular carcinoma cells implanted nude mice model.

Tumor angiogenesis is an important process for several kinds of tumor, especially, during its angiogenic switch. The present study was aimed to investigate the dynamical process of tumor neocapillarization in Hepatocellular carcinoma cells implanted nude mice model. Male BALB/c nude mice (20-25 g) were used. After the implantation of a dorsal skin-fold chamber, the Hepatocellular carcinoma cells (HepG2) (30 microl of 2 x 10(6) cells) were inoculated into the upper layer of the skin within the chamber (HepG2-group), while the shammed control group (Sham-group) was received a normal saline. Intravital fluorescence videomicroscopy was performed to monitor the tumor neocapillary on days 7 and 14 post-inoculation by intravenous injection of 0.1 ml rhodamine B isothiocyanate-labeled dextran (0.5%). Based on the recorded videoimages, the tumor microvascular networks were measured using a digital image analysis. The neovascular density and configuration were represented in terms of microvascular density index (MVDI) and neovascular bifurcation ratio (BR). The MVDI levels of HepG2-group were significantly increased on day 7 and 14 compared to the age-matched Sham-group. The increase in MVDI agreed with the increase in the BR. In conclusion, our dorsal skin-fold chamber technique with intravital fluorescence videomicroscopy and digital image analysis was most useful to monitor the neovascular network for quantitatively evaluating the progression of tumor angiogenesis in terms of MVDI and BR values.

Preventive mechanism of genistein on coronary endothelial dysfunction in ovariectomized rats: an isolated arrested heart model.

The effects of genistein on coronary endothelial dysfunction in bilateral ovariectomized rats were examined. Female Wistar rats were subjected to a bilateral ovariectomy (OVX rat). The animals were divided into three groups: sham treated with vehicle (DMSO 100 microl/day, Sham-DMSO), OVX treated with vehicle (DMSO 100 microl/day, OVX-DMSO), and OVX treated with genistein (0.25 mg/kgBW/day, OVX-genistein). Mean arterial pressure (MAP), heart rate (HR), body weight (BW), uterine weight and plasma E2 were monitored at 4- and 10-week after the treatment. We investigated the endothelium-dependent and -independent vasorelaxation by using acetylcholine (Ach 10(-5) M) and sodium nitroprusside (SNP 10(-7) M), respectively. The experimental results indicated that the uterine weights of all OVX rats were significantly decreased as compared to the sham groups. HR and MAP of both OVX-DMSO and OVX-genistein on 4 and 10 weeks were no significantly increased as compared to the Sham groups. The present coronary vasodilatation responses demonstrated only the significant decrement of endothelium-dependent, not for endothelium-independent, in OVX rats. The treatment of genistein could significantly attenuate this abnormality (% changes of vessel diameter obtained after Ach 10(-6) M: Sham-DMSO(10-wk) = 10.96 +/- 1.2%, OVX-DMSO(10-wk) = 3.2 +/- 0.77%, OVX-genistein(10-wk) = 11.45 +/- 1.85%), (% changes of vessels diameter obtained after SNP 10(-7)M: Sham-DMSO(10-wk) = 16.05 +/- 2.82%, OVX-DMSO(10-wk) = 12.73 +/- 2.72%, OVX-genistein(10-wk) = 16.4 +/- 4.71%) (p < 0.05). However, the lipid profiles monitored from all groups of 4 and 10 weeks did not demonstrate any significant changes. Therefore, it implied that endothelial dysfunction was not primarily cause by the lipid profiles changing in ovariectomized rats. Moreover, such effects of estrogen lacking on coronary endothelial-dependent vasodilatation could be attenuated by genistein supplementation. The present findings suggest that genistein might be used as an therapeutic agent for preventing the menopausal vascular complications.

Effects of Aloe vera on leukocyte adhesion and TNF-alpha and IL-6 levels in burn wounded rats.

The effects of Aloe vera on microcirculation and levels of TNF-alpha and IL-6 were investigated in rats after inducing burn. Seventy-two male Wistar Furth rats were equally divided into four groups as follow: controls (CON), untreated burn-wound rats (BURN), normal saline-treated burn-wound rats (BURN-NSS) and Aloe vera-treated burn-wound rats (BURN-ALOE). The animals in each group were equally subdivided into three subgroups for the study on day 3, 7 and 14 post-burn. Dorsal skinfold chamber preparation and intravital fluorescence microscopic technique were performed to examine leukocyte adhesion on postcapillary venules. ELISA techniques were performed to examine serum TNF-alpha and IL-6 levels. It was found that the amount of leukocyte adhesion was significantly reduced in the BURN-ALOE group compared to rats in the BURN group on day 14. Levels of TNF-alpha and IL-6 were also decreased significantly compared to BURN at all three monitored time points. Aloe vera could inhibit the inflammatory process following burn injury, as characterized by the reduction of leukocyte adhesion, as well as those pro-inflammatory cytokines.

Genistein replacement therapy for vasodilation disorder in bilateral ovariectomized rats.

The objective of this study is to examine the effects of genistein on endothelial dysfunction in bilateral ovariectomized rats. Female Wistar rats were subjected to a bilateral ovariectomy (OVX rat). The animals were divided into three groups: sham treated with vehicle (DMSO 100 microliters/day, Shamveh), OVX treated with vehicle (DMSO 100 microliters/day, OVXveh), and OVX treated with genistein (0.25 mg/kg BW/day, OVXgen). Mean arterial pressure (MAP), heart rate (HR), body weight (BW), uterine weight and plasma E2 were monitored at 4-week after the treatment. We investigated the endothelium-dependent and -independent vasorelaxation by using acetylcholine (Ach 10(-6) M) and sodium nitroprusside (SNP 10(-7) M), respectively. The experimental results indicated that the uterine weights of all OVX rats were significantly decreased as compared to the sham groups (OVXveh = 0.007+/-0.004 g, OVXgen =0.003+/-0.001 g, Shamveh =0.017+/-0.001 g). MAP of OVXveh group was significantly increased compared to the Sham group (OVXveh=139.99+/-7.50 mmHg, Shamveh =118.10+/-19.33 mmHg, p<0.05). No significant increase in MAP was observed in OVXgen (OVXgen =123.33+/-8.61 mmHg; p<0.05). HR showed no significant difference among those groups. The present study of vasodilator responses demonstrated only the significant decrease in endothelium-dependent, not for endothelium-independence, in OVX rats, while the treatment of genistein could significantly attenuate this abnormality (OVXveh =3.03+/-3.99%, Shamveh =45.46+/-3.59%, OVXgen =33.52+/-3.25% in % change of vessel diameter). The present findings suggest that genistein could be used as a therapeutic agent for menopausal vascular complications.

Endothelial nitric oxide synthase expression in systemic and pulmonary circulation of streptozotocin induced diabetic rats: comparison using image analysis.

To compare the level of endothelial nitric oxide synthase (eNOS) expression produced in heart and lung vascular tissue, the protein content was determined using Western blot analysis with the enhancement of image processing. Heart and lung extracts from 12 and 24 weeks from control (CON) and streptozotocin-induced diabetic (DM) rats were collected for Western blot analysis. Using monoclonal antibody against rat eNOS protein (140 kDa), the eNOS-protein bands were detected with enhanced chemiluminescence (ECL; Amersham) and exposured to film (Hyperfilm-ECL; Amersham). Images of eNOS bands on each film were then scanned and saved to digital files. Using Global Lab Image software, the number of pixels in each digital file was counted and calibrated for eNOS-protein content. For the CON and DM groups, the mean values of eNOS-protein contents were calculated and expressed as a percentage of total protein content, 5 micrograms. It was found that the eNOS level in DM hearts was significantly decreased, as compared to age-matched CON hearts. On the other hand, eNOS levels in DM lungs was increased, compared to CON lungs. Therefore, it may be concluded that high, not low, flow-mediated eNOS expression is a good measure of hyperglycemic-induced endothelial dysfunction.

Effects of ACE-I on diabetic cardiovascular complications: anti-hypertensive and non-antihypertensive doses.

To evaluate the effects of angiotensin converting enzyme inhibitor (ACE-I) on diabetic cardiovascular complications, a streptozotozin (STZ, i.p., 70 mg/kg BW) induced diabetes rat model was used. The animals were separated into four major groups including: control (NSS), STZ-treated rats, STZ-treated rats received daily oral feeding of cilazapril starting one day after STZ injection (STZ-C1), and STZ-treated rats received daily oral feeding of cilazapril eight weeks after the STZ injection (STZ-C8). Within the groups of STZ-C1 and STZ-C8, the animals were also divided into three subgroups of six rats that received different doses of cilazapril treatment, 0.01 mg/Kg BW, 1 mg/Kg BW, and 10 mg/Kg BW. By using the modified isolated heart model, the parameters of mean arterial pressure (MAP), heart rate (HR), left ventricular isotonic contraction (LVIC), aortic flow rate (AFR), coronary flow rate (CFR), and ratio of heart weight per body weight (R) were assessed for each groups 8 and 20 weeks after the STZ injections. Moreover, the changes of wall thickness of the left ventricular wall (LV), right ventricular wall (RV), and interventricular septal wall (IVS) were monitored from the scanning electron micrographs of each heart. The results indicated that in both STZ-C1 and STZ-C8, the diabetic hypertension could be prevented or treated by anti-hypertensive doses of cilazaprils. Besides, the values of AFR, CFR, and LVIC were significantly increased when comparing between the STZ and STZ-C1 or STZ-C8. The results of morphological examinations indicated that: (1) left ventricular walls of the three hearts of STZ-rats had increased significantly more than controls. (2) Right ventricular walls and interventricular septal walls were not significantly different among STZ-rats, cilazapril treated STZ-rats and age matched controls. Therefore, it is concluded that ACE-I could act either as a cardioprotective or therapeutic agent for diabetic hearts. Both major anti-hypertension and anti-trophic effects of ACE-I have already been elucidated.

Effect of serotonin depletion on nitric oxide induced cerebrovascular nociceptive response.

The study was conducted to investigate the effect of serotonin depletion on nitric oxide-induced meningeal vascular response and cerebrovascular nociception. Nitroglycerin was infused i.v. to control and serotonin-depleted rats. Pial circulation was monitored by intravital fluorescent videomicroscopy and Fos immunoreactivity trigeminal nucleus caudalis neurons was used as an indicator for the cerebrovascular nociception. The results showed that the degree of nitric oxide-induced pial microvascular dilatation was significantly greater in the serotonin-depleted group than the control. The number of nitric oxide-evoked Fos-immunoreactive cells between the two groups remained comparable. The results suggest that though depletion of serotonin can facilitate the vascular response to nitric oxide it does not alter the nitric oxide-induced craniovascular nociceptive response.

Comparative effects of garlic and aspirin on diabetic cardiovascular complications.

By using streptozotocin-induced diabetic rats as a studied model, our previous experimental results have indicated that daily oral feeding of garlic extract (100 mg/kg BW) could increase the cardiovascular functions in streptozotocin (STZ) rats; the abnormality of lipid profile was prevented; and garlic extract could increase fibrinolitic activities with the decrease of platelet aggregation. Moreover, the plasma insulin level was increased concomitantly with the decrease of plasma glucose level. However, due to the high incidence of atherosclerosis in diabetes, the present study has been continued for further investigation of the effect of garlic extract on the coronary vascular ultrastructural changes. In addition, to identify the possible mechanism(s) of garlic's therapeutic effects, the cyclooxygenase inhibitor, aspirin, has been included in this present study. By using transmission electron microscopic studies, 16 weeks of daily oral feeding of garlic extract (100 mg/kg BW) caused as an antiatherosclerotic agent at the coronary arteriolar (15-30 microns) wall in STZ-rats. Interestingly, the thickening of coronary capillary (5-10 microns) basement membrane also was significantly attenuated within the group of STZ-rats treated with garlic extract. However, the possible direct action of garlic through the cyclooxygenase pathway has not been confirmed by the results of aspirin: The daily oral feeding of aspirin (10 mg/kg BW) in 16-week STZ-rats has not showed reduced arteriolar vascular wall abnormalities. The irregular patterns of fiber matrix, arranging the basement membrane at the arteriolar walls, were still recognized in the same manner as in STZ-rats. Interestingly, the thickening of the capillary basement membrane occurred in 16-week STZ-rats seems to be attenuated by the aspirin received. At present, garlic extract may open the new era in the medicinal use of garlic to prevent diabetic cardiovascular complications.

Therapeutic effects of Aloe vera on cutaneous microcirculation and wound healing in second degree burn model in rats.

OBJECTIVE: To demonstrate the microcirculatory and wound healing effects of Aloe vera on induced second degree burn wounds in rats. METHOD: A total of 48 male Wistar rats were equally divided into 4 groups as follows: sham controls, untreated burn-wound rats, those treated with once-daily application of normal saline (NSS) and those treated with once-daily application of lyophilized Aloe vera gel. The animals in each group were equally subdivided into 2 subgroups for the study of cutaneous microcirculation and wound healing on day 7 and 14 after burn. Dorsal skinfold chamber preparation and intravital fluorescence microscopic technique were performed to examine dermal microvascular changes, including arteriolar diameter, postcapillary venular permeability and leukocyte adhesion on postcapillary venules. RESULTS: On day 7, the vasodilation and increased postcapillary venular permeability as encountered in the untreated burn were found to be reduced significantly (p < 0.05) in both the NSS- and Aloe vera-treated groups, but to a greater extent in the latter. Leukocyte adhesion was not different among the untreated, NSS- and Aloe vera-treated groups. On day 14, vasoconstriction occurred after the wound had been left untreated. Only in the Aloe vera-treated groups, was arteriolar diameter increased up to normal condition and postcapillary venular permeability was not different from the sham controls. The amount of leukocyte adhesion was also less observed compared to the untreated and NSS- treated groups. Besides, the healing area of the Aloe vera-treated wound was better than that of the untreated and NSS- treated groups during 7 and 14 days after burn. CONCLUSION: Aloe vera could exhibit the actions of both anti-inflammation and wound healing promotion when applied on a second degree burn wound.

Enhanced tumor necrosis factor in the serum and renal hypoperfusion in nephrosis associated with focal segmental glomerulosclerosis.

Enhanced tumor necrosis factor alpha associated with immunocirculatory imbalance expressed as increased ratio between proinflammatory (TNFalpha) and antiinflammatory (IL-10) cytokines was observed in the serum of nephrosis associated with focal segmental glomerulosclerosis. Such altered immunocirculatory balance correlated with the reduction in renal plasma flow determined by the intrarenal hemodynamic study by which it implies that a glomerular endothelial cell injury associated with impaired renal perfusion is likely to be spontaneously induced by enhanced tumor necrosis factor in the presence of inadequate release of antiinflammatory cytokine (IL-10).

Hyposerotonin-induced nitric oxide supersensitivity in the cerebral microcirculation.

OBJECTIVE: To investigate the relationship between hyposerotonin and cranial microvascular responses to nitric oxide (NO). BACKGROUND: Although the mechanism underlying NO supersensitivity in migraine is still unclear, an alteration of the serotonin system is a possible explanation. METHODS: Wistar rats were divided into control and hyposerotonin groups. Serotonin was depleted by intraperitoneal injection with 300 mg/kg of para-chlorophenylalanine (PCPA), a tryptophan hydroxylase inhibitor. Three days after PCPA pretreatment, the animals were prepared for assessment of their NO-induced vasomotor response using glyceryl trinitrate (GTN: 8 to 10 mg/kg, intravenously) as an NO donor. Pial circulation was visualized by the intravital fluorescein videomicroscopic technique. Images of vessels at 0, 5, 15, 30, and 60 minutes post GTN infusion were digitized and measured. At the end of monitoring, the rat brains were removed for ultrastructural study of the brain microvessels. RESULTS: Infusion of GTN produced dose-dependent pial arteriolar dilatation. This vasodilator effect was significantly increased in the PCPA-treated groups, especially at 30 and 60 minutes. The percentage change from baseline diameter at 30 minutes after the 8-mg/kg GTN infusion was 42.6 +/- 3.1 for the hyposerotonin group and 16.8 +/- 2.9 for the control group (P<.001). Electron microscopic study revealed that exposure to the NO donor produced considerable changes in cerebral microvessels, characterized by focal ballooning of endothelial cells, increased microvillous formation, and increased endothelial pinocytosis. These anatomical changes were significantly more prominent in the hyposerotonin group. CONCLUSIONS: A hyposerotoninergic condition can facilitate the NO-induced physiological and pathological responses in meningeal and cerebral microvessels and, therefore, is a possible explanation for the supersensitivity to NO observed in patients with migraine.

Effects of adrenomedullin on the cardiac performance and coronary flow in an isolated perfused rat heart model.

The effect of adrenomedullin (AM) on the cardiac performance and coronary flow were studied in an isolated perfused rat heart model based on the modified Langendorff method. The heart rate (HR), electrocardiogram (ECG), left ventricular contraction (LVC) (dP/dt), and coronary flow (CF) were measured before and after the application of AM. The effect of AM on the coronary flow was examined in the model with and without endothelial degradation, using different inhibitors such as N(G)-nitro-L-arginine, glibenclamide, and indomethacin. The present results indicated that AM increased HR and CF, but decreased LVC significantly, while it had no effect on ECG. The vasodilatory effect of AM was discussed in views of endothelial-dependence due to nitric oxide and K+ channel activation.