Relation of Helicobacter pylori infection to peripheral arterial stiffness and 10-year cardiovascular risk in subjects with diabetes mellitus.

PURPOSE: This study was conducted to investigate the relation of HP infection to peripheral arterial stiffness and 10-year cardiovascular risk in diabetes mellitus (DM). METHODS: DM subjects who underwent the C13-breath test were enrolled and divided into DMHP+ and DMHP- groups. Peripheral arterial stiffness was measured using brachial to ankle pulse wave velocity (baPWV). Framingham score (FRS) and Chinese evaluation method of ischemic cardiovascular diseases (ICVD) were used to clarify 10-year cardiovascular risk. RESULTS: A total of 6767 subjects were included, baPWV and proportion of subjects with severe peripheral arterial stiffness were lower in DMHP- group than DMHP+ group (1556.68 ± 227.54 vs 2031.61 ± 525.48 cm/s, p < 0.01; 21.9% vs 62.7%, p < 0.01). Multivariate logistic regression analysis demonstrated that HP infection was independently associated with baPWV. Furthermore, cardiovascular risk score and the proportion of subjects with high risk were lower in DMHP- group than DMHP+ group (FRS: 12.09 ± 3.77 vs 13.91 ± 3.77, 17.2% vs 38.8%; ICVD: 8.56 ± 2.99 vs 10.22 ± 3.16, 43.9% vs 65.4%, with all p < 0.05). CONCLUSION: DM subjects with HP infection had more severe peripheral arterial stiffness compared those without HP infection, a higher cardiovascular risk score and 10-year cardiovascular risk stratification were observed in those subjects.

Clinical Study on Prelaryngeal Lymph Node Metastasis in Papillary Thyroid Carcinoma.

OBJECTIVE: This study aims to investigate the risk factors of prelaryngeal lymph node metastasis in papillary thyroid carcinoma and its clinical application value. METHODS: The clinical pathological features and metastatic risks were statistically analyzed by reviewing 254 patients with papillary thyroid carcinoma, who received their first operation and prelaryngeal lymph node dissection in our department. RESULTS: The detection of prelaryngeal lymph nodes, tumor size and any paratracheal lymph node metastasis were correlated with the number of paratracheal lymph node metastasis (P<0.05), but these were not correlated with age, gender, multiple foci, tumor size, any paratracheal lymph node metastasis, metastatic location, or foci location (P>0.05). CONCLUSION: Paratracheal lymph node metastasis indicates a high possibility of prelaryngeal lymph node metastasis. Paratracheal lymph node dissection combined with prelaryngeal lymph node dissection should be simultaneously considered in operations for thyroid papilla carcinoma.

[Effect of catgut implantation at "Yingxiang"(LI20) on lower airway remodeling in allergic rhinitis rats].

OBJECTIVE: To observe the effect of catgut implantation at "Yingxiang"(LI20) on lower airway remodeling and levels of osteopon-tin (OPN) protein in allergic rhinitis (AR) rats, so as to reveal its mechanisms underlying improvement of AR. METHODS: Male SD rats were randomly divided into control, model and catgut implantation groups, with 10 rats in each group. The AR model was established by intraperitoneal injection and nasal drip of ovalbumin. The catgut implantation was applied to bilateral "Yingxiang"(LI20) for 28 days in rats of the catgut implantation group. The total score of allergic symptoms of rats in each group were observed. The histopathological changes of lower airway were observed under light microscope after Hematoxylineosin, Periodic acid-Schiff and Masson staining. The expression of OPN protein was detected by immunohistochemistry and Western blot, separately. RESULTS: The total score of allergic symptoms of nose-wiping, running nose and sneezing, count of lung goblet cells, lung fiber content, and immunoactivity and expression levels of OPN protein were significantly increased in the model group in contrast to the control group (P<0.05). After the intervention, the total score of allergic symptoms, count of lung goblet cells, immunoactivity and expression levels of OPN protein were considerably down-regulated in the catgut implantation group relevant to the model group (P<0.05). H．E. stain showed thickening of partial airway wall, narrowing of lumen, increase of mucus section, widened alveolar septum, infiltration of inflammatory cells, lymphocytes and eosinophil around the bronchus and in the lung interstitium in AR rats, which was milder in the catgut implantation group. The immunoactivity and expression levels of OPN protein were positively related with the lung goblet cells count and lung fiber content (P<0.05，P<0.01). CONCLUSION: Acupoint implantation of catgut can improve pathological changes of lower airway remodeling, which may be related to its effect in down-regulating the expression of OPN protein in the lung tissue.

Relationship between plasma levels of 25-hydroxyvitamin D and arterial stiffness in elderly Chinese with non-dipper hypertension: An observational study.

Elderly individuals with non-dipper hypertension are at high risk of cardiovascular disease because of increased stiffness of peripheral arteries. Since, vitamin D deficiency is prevalent in elderly Chinese. We examined whether reduced plasma levels of 25-hydroxyvitamin D [25(OH)D] may help promote this stiffness.Hypertensive patients at least 60 years old without history of peripheral arterial disease at our hospital were retrospectively divided into dipper and non-dipper groups according to the results of 24-hour ambulatory blood pressure monitoring. Plasma levels of 25(OH)D were measured by enzyme immunoassay. Peripheral arterial stiffness was measured based on the cardio-ankle vascular index (CAVI).Of the 155 patients enrolled, 95 (61.3%) were diagnosed with non-dipper hypertension and these patients had significantly lower plasma levels of 25(OH)D than the 60 patients with dipper hypertension (19.58 ±â€Š5.97 vs 24.36 ±â€Š6.95 nmol/L, P < .01) as well as significantly higher CAVI (8.46 ±â€Š1.65 vs 7.56 ±â€Š1.08 m/s, P < .01). Vitamin D deficiency was significantly more common among non-dipper patients (57.9% vs 31.7%, P < .01). Multivariate regression showed that age and 25(OH)D were independently related to CAVI, with each 1-ng/ml decrease in 25(OH)D associated with a CAVI increase of +0.04 m/s.Non-dipper hypertension is associated with vitamin D deficiency and reduced plasma levels of 25(OH)D. The latter may contribute to stiffening of peripheral arteries, increasing the risk of cardiovascular disease.

Vascular endothelial growth factor receptor inhibitor enhances dietary salt-induced hypertension in Sprague-Dawley rats.

Clinical evidence links the inhibition of VEGF to hypertension. However, the mechanisms by which VEGF affects the pathogenesis of hypertension remain in question. We determined 1) whether administration of VEGF receptor inhibitor SU5416 enhances dietary salt-induced hypertension in Sprague-Dawley (SD) rats, and 2) whether VEGF or SU5416 directly affects proliferation of cultured human renal proximal tubular epithelial cells (HRPTEC) and endothelial nitric oxide synthase (eNOS) expression in cultured human glomerular microvessel endothelial cells (HGMEC). Ten 10-wk-old male SD rats received a high sodium diet (HS; 8%) and the other 10 SD rats received a normal sodium diet (NS; 0.5%) for 4 wks. After 2 wks of the dietary program, five rats were administered with SU5416 at 10 mg x kg(-1) x day(-1) ip or DMSO (vehicle) for 14 days in HS and NS groups. Mean arterial pressure was significantly higher in rats treated with SU5416, as opposed to those treated with DMSO and fed with HS for 4 wk (157.6 +/- 3.9 vs. 125.9 +/- 4.3 mmHg, P < 0.01). Increased proteinuria and albuminuria were associated with marked renal histological abnormalities in HS group with SU5416 administration, compared with those in the vehicle HS group. 3H-thymidine incorporation assay showed that SU5416 blocked the actions of both exogenous and endogenous VEGF on the proliferation of HRPTEC. VEGF (10 ng/ml) significantly increased eNOS protein levels by 29% in cultured HGMEC, but its action was completely abolished by SU5416. These results suggest that VEGF receptor inhibition enhances dietary salt-induced hypertension and kidney injury, possibly by direct damage on renal cells and decreasing NO production by eNOS.

Long-term high salt diet causes hypertension and alters renal cytokine gene expression profiles in Sprague-Dawley rats.

OBJECTIVE: The present study examines whether a long-term high salt diet causes hypertension and renal injury in normal subjects [Sprague-Dawley (SD) rats] and alters renal cytokine-related gene expression profiles. METHODS: Four 10 week old male SD rats received a high salt diet (HS, 8%) and the other 4 SD rats received a normal salt diet (NS, 0.5%) for 8 weeks. Mean arterial pressure (MAP) and renal damages such as albuminuria and histological renal injury were determined. The relative mRNA levels of 514 cytokine-related genes (normalized by beta-actin) in rat kidneys following NS or HS were determined quantitatively through analysis of 4 sets of gene expression profiles using the mouse cDNA membrane microarrays. RESULTS: We demonstrated that 8 weeks of HS diet increased MAP [(140.0+/-5.3) vs (112.0+/-2.2) mmHg; 1 mmHg=0.133 kPa, P<0.01], albuminuria [(41.4+/-3.2) vs (20.1+/-4.5) mg/d; P<0.01], and caused histological renal injury in SD rats, compared to NS group. Of the 514 genes in the array, there were 27 (5.25%) genes with significantly different expression in the kidney of SD rats with HS compared to those of SD rats with NS. Functional clustering analysis indicated the following functional pathways related to high salt diet-induced hypertension: (1) pro-inflammatory response ( upward arrowIL-17, CCL28; downward arrow NFkappabib); (2) endothelial dysfunction ( downward arrowVEGF-A, VEGF-B, endoglin); (3) pro-matrix formation ( upward arrowosteopontin, IGFBP-5; downward arrow IFN-gamma); and (4) attenuated cell survival and differentiation ( downward arrowCNTF, IGF-II R, ephrin-B1). Northern blot confirmed that 8 weeks of HS diet significantly decreased renal expression of VEGF mRNA, compared to NS group (P<0.01). ELISA showed that HS diet significantly decreased renal protein levels of VEGF and CCL28. CONCLUSION: These findings support the hypothesis that hypertension can be induced in normal rats by a long-term high salt diet, which is associated with increased renal injury and marked changes in renal cytokine gene expression profiles that are closely related to the pro-inflammatory response, pro-matrix formation, endothelial dysfunction, and attenuated cell survival and differentiation.

Adenosine infusion increases plasma levels of VEGF in humans.

BACKGROUND: Many in vitro studies have shown that adenosine (Ado) can induce vascular endothelial growth factor (VEGF) mRNA and protein expression and stimulate endothelial proliferation. In the present study, we seek to determine whether Ado can increase circulating levels of VEGF protein in the intact human. METHODS: Five outpatients 49.3 +/- 6.7 years of age and weighing 88.2 +/- 8.5 kg were selected. They were given a 6 min intravenous infusion of Ado (0.14 mg kg-1 min-1) in conjunction with sestamibi myocardial perfusion scans. Mean blood pressure (MBP, calculated from systolic and diastolic values) and heart rate (HR) were determined before Ado infusion and every 2 min for the next 10 min. Plasma VEGF concentrations (ELISA) were determined immediately before Ado infusion and 1 h, 2 h, and 8 h after the infusion. RESULTS: Plasma VEGF concentration averaged 20.3 +/- 2.0 pg ml-1 prior to Ado infusion, and increased to 62.7 +/- 18.1 pg ml-1 at 1 h post- infusion (p < 0.01). VEGF plasma concentration returned to basal levels 2 h after infusion (23.3 +/- 3.4 pg ml-1). MBP averaged 116 +/- 7 mmHg and heart rate averaged 70 +/- 7 prior to Ado infusion. MBP decreased by a maximum of approximately 22% and HR increased by a maximum of approximately 17% during the infusion. CONCLUSION: We conclude from these preliminary findings that intravenous infusion of adenosine can increase plasma levels of VEGF in humans.

Exercise increases endostatin in circulation of healthy volunteers.

BACKGROUND: Physical inactivity increases the risk of atherosclerosis. However, the molecular mechanisms of this relation are poorly understood. A recent report indicates that endostatin, an endogenous angiostatic factor, inhibits the progression of atherosclerosis, and suggests that reducing intimal and atherosclerotic plaque tissue neovascularization can inhibit the progression atherosclerosis in animal models. We hypothesize that exercise can elevate the circulatory endostatin level. Hence, exercise can protect against one of the mechanisms of atherosclerosis. RESULTS: We examined treadmill exercise tests in healthy volunteers to determine the effect of exercise on plasma levels of endostatin and other angiogenic regulators. Oxygen consumption (VO2) was calculated. Plasma levels of endostatin, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were determined using ELISA. The total peak VO2 (L) in 7 male subjects was 29.5 +/- 17.8 over a 4-10 minute interval of exercise. Basal plasma levels of endostatin (immediately before exercise) were 20.3 +/- 3.2 pg/ml, the plasma levels increased to 29.3 +/- 4.2, 35.2 +/- 1.8, and 27.1 +/- 2.2 ng/ml, at 0.5, 2, and 6 h, respectively, after exercise. There was a strong linear correlation between increased plasma levels of endostatin (%) and the total peak VO2 (L) related to exercise (R2 = 0.9388; P < 0.01). Concurrently, VEGF levels decreased to 28.3 +/- 6.4, 17.6 +/- 2.4, and 26.5 +/- 12.5 pg/ml, at 0.5, 2, and 6 h, respectively, after exercise. There were no significant changes in plasma bFGF levels in those subjects before and after exercise. CONCLUSIONS: The results suggest that circulating endostatin can be significantly increased by exercise in proportion to the peak oxygen consumption under physiological conditions in healthy volunteers. These findings may provide new insights into the molecular links between physical inactivity and the risk of angiogenesis dependent diseases such as atherosclerosis.

Sunitinib significantly suppresses the proliferation, migration, apoptosis resistance, tumor angiogenesis and growth of triple-negative breast cancers but increases breast cancer stem cells.

The majority of triple-negative breast cancers (TNBCs) are basal-like breast cancers. However there is no reported study on anti-tumor effects of sunitinib in xenografts of basal-like TNBC (MDA-MB-468) cells. In the present study, MDA-MB-231, MDA-MB-468, MCF-7 cells were cultured using RPMI 1640 media with 10% FBS. Vascular endothelia growth factor (VEGF) protein levels were detected using ELISA (R & D Systams). MDA-MB-468 cells were exposed to sunitinib for 18 hours for measuring proliferation (3H-thymidine incorporation), migration (BD Invasion Chamber), and apoptosis (ApopTag and ApoScreen Anuexin V Kit). The effect of sunitinib on Notch-1 expression was determined by Western blot in cultured MDA-MB-468 cells. 10(6) MDA-MB-468 cells were inoculated into the left fourth mammary gland fat pad in athymic nude-foxn1 mice. When the tumor volume reached 100 mm(3), sunitinib was given by gavage at 80 mg/kg/2 days for 4 weeks. Tumor angiogenesis was determined by CD31 immunohistochemistry. Breast cancer stem cells (CSCs) isolated from the tumors were determined by flow cytometry analysis using CD44(+)/CD24(-) or low. ELISA indicated that VEGF was much more highly expressed in MDA-MB-468 cells than MDA-MB-231 and MCF-7 cells. Sunitinib significantly inhibited the proliferation, invasion, and apoptosis resistance in cultured basal like breast cancer cells. Sunitinib significantly increased the expression of Notch-1 protein in cultured MDA-MB-468 or MDA-MB-231 cells. The xenograft models showed that oral sunitinib significantly reduced the tumor volume of TNBCs in association with the inhibition of tumor angiogeneisis, but increased breast CSCs. These findings support the hypothesis that the possibility should be considered of sunitinib increasing breast CSCs though it inhibits TNBC tumor angiogenesis and growth/progression, and that effects of sunitinib on Notch expression and hypoxia may increase breast cancer stem cells. This work provides the groundwork for an innovative therapeutic strategy in TNBC therapy by using sunitinib plus γ-secretase inhibitor to simultaneously target angiogenesis and CSC.

EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression.

The role of EGCG, a major green tea catechin in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Sixteen eight-wk-old female mice (C57BL/6 J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. Eight mice received EGCG at 50-100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. EGCG treatment significantly reduced tumor weight over the control (0.37 ± 0.15 vs. 1.16 ± 0.30 g; P < 0.01), tumor CD (109 ± 20 vs. 156 ± 12 capillary #/mm^2; P < 0.01), tumor VEGF expression (45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; P < 0.01), respectively. But, it has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. EGCG at 50 µg/ml significantly inhibited the activation of HIF-1α and NFκB as well as VEGF expression in cultured E0771 cells, compared to the control, respectively. These findings support the hypothesis that EGCG, a major green tea catechin, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression.

Increased plasma levels of soluble vascular endothelial growth factor receptor 1 (sFlt-1) in women by moderate exercise and increased plasma levels of vascular endothelial growth factor in overweight/obese women.

The incidence of breast cancer is increasing worldwide, and this seems to be related to an increase in lifestyle risk factors, including physical inactivity and overweight/obesity. We have reported previously that exercise induced a circulating angiostatic phenotype characterized by increased soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin and decreased unbound vascular endothelial growth factor (VEGF) in men. However, there are no data on women. The present study determines the following: (a) whether moderate exercise increased sFlt-1 and endostatin and decreased unbound VEGF in the circulation of adult female volunteers and (b) whether overweight/obese women have a higher plasma level of unbound VEGF than lean women. A total of 72 African American and White adult women volunteers ranging in age from 18 to 44 years were enrolled in the exercise study. All the participants walked on a treadmill for 30 min at a moderate intensity (55-59% heart rate reserve), and oxygen consumption (VO(2)) was quantified utilizing a metabolic cart. We obtained blood samples before and immediately after exercise from 63 participants. ELISA assays showed that the plasma levels of sFlt-1 were 67.8±3.7 pg/ml immediately after exercise (30 min), significantly higher than the basal levels, 54.5±3.3 pg/ml, before exercise (P<0.01; n=63). There was no significant difference in the % increase in the sFlt-1 levels after exercise between African American and White (P=0.533) women or between lean and overweight/obese women (P=0.892). There was no significant difference in the plasma levels of unbound VEGF (35.28±5.47 vs. 35.23±4.96 pg/ml; P=0.99) or endostatin (111.12±5.48 vs. 115.45±7.15 ng/ml; P=0.63) before and after exercise. The basal plasma levels of unbound VEGF in overweight/obese women were 52.26±9.6 pg/ml, significantly higher than the basal levels of unbound VEGF in lean women, 27.34±4.99 pg/ml (P<0.05). The results support our hypothesis that exercise-induced plasma levels of sFlt-1 could be an important clinical biomarker to explore the mechanisms of exercise training in reducing the progression of breast cancer and that VEGF is an important biomarker in obesity and obesity-related cancer progression.

Notch signals in the endothelium and cancer "stem-like" cells: opportunities for cancer therapy.

Anti-angiogenesis agents and the identification of cancer stem-like cells (CSC) are opening new avenues for targeted cancer therapy. Recent evidence indicates that angiogenesis regulatory pathways and developmental pathways that control CSC fate are intimately connected, and that endothelial cells are a key component of the CSC niche. Numerous anti-angiogenic therapies developed so far target the VEGF pathway. However, VEGF-targeted therapy is hindered by clinical resistance and side effects, and new approaches are needed. One such approach may be direct targeting of tumor endothelial cell fate determination. Interfering with tumor endothelial cells growth and survival could inhibit not only angiogenesis but also the self-replication of CSC, which relies on signals from surrounding endothelial cells in the tumor microenvironment. The Notch pathway is central to controlling cell fate both during angiogenesis and in CSC from several tumors. A number of investigational Notch inhibitors are being developed. Understanding how Notch interacts with other factors that control endothelial cell functions and angiogenesis in cancers could pave the way to innovative therapeutic strategies that simultaneously target angiogenesis and CSC.

Postmenopausal obesity promotes tumor angiogenesis and breast cancer progression in mice.

Obese postmenopausal women have a 50% higher risk of breast cancer than non-obese women. There is not an animal model that mimics postmenopausal obesity related to breast cancer progression. Using age-relevant C57BL/6 mice, this study determined whether postmenopausal obesity increases VEGF expression, tumor angiogenesis, and breast tumor growth. Ovariectomy (OVX) was performed in 12 sixty week-old female mice, then followed by a low-fat (5%, LF, n=6) or a high-fat (60%, HF, n=6) diet for 12 weeks. In the eighth week of the dietary program, 10(6) E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored for 4 weeks. Body weights were monitored weekly. At the end of the experiment, blood samples, visceral fat and tumors were collected for measuring VEGF expression using ELISA and intratumoral microvessel density (IMD) using CD31 immunochemistry. Body weight was significantly increased in OVX/HF mice, compared to OVX/LF group (55.3±1.7 vs. 41.5±1.5 g; p < 0.01). There was a two-fold increase in the ratio of visceral fat/BW in OVX/HF mice, compared to those in OVX/LF group (0.062±0.005 vs. 0.032±0.003; p < 0.01). Postmenopausal obesity significantly increased breast tumor weight over the control (4.62±0.63 vs. 1.98±0.27 g; p < 0.01) and IMD (173±3.7 vs. 139±4.3 IM#/mm^2; p < 0.01). Tumor VEGF levels were higher in OVX/HF mice, compared to OVX/LF group (73.3±3.8 vs. 49.5±4.3 pg/mg protein; p < 0.01). Plasma VEGF levels (69±7.1 vs. 48±3.5 pg/ml) and visceral fat VEGF levels (424.4±39.5 vs. 208.5±22.4 pg/mg protein) were significantly increased in OVX/HF mice, compared to OVX/LF group, respectively (n=6; p < 0.01). Interestingly, adipose tissue primary culture showed that subcutaneous fat released more VEGF, compared to visceral fat (6.77±1.14 vs. 0.94±0.16 pg/mg tissue; n=6; p < 0.01). These findings support the hypothesis that postmenopausal obesity promotes tumor angiogenesis and breast cancer progression, possibly through increased adipose tissue mass and adipokines such as VEGF that could systemically and locally affect breast cancer progression.

SU11248, a selective tyrosine kinases inhibitor suppresses breast tumor angiogenesis and growth via targeting both tumor vasculature and breast cancer cells.

SU11248 is a selective inhibitor of certain protein kinases including VEGFR types 1-3 that are expressed in human breast cancer. The present study determines whether the anti-tumor activity of SU11248 results from the inhibition of angiogenesis, as well as direct anti-proliferation and anti-migration effects on breast tumors. Eight-wk old female mice (C57BL/6) were given SU11248 at 20-40 mg/kg/d in drinking (distilled) water for 4 wks. Control mice received drinking water only. In the 2nd wk, 10(6) E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored using dial calipers. At the end, tumors were isolated for measuring tumor size and intratumoral microvessel density (IMD) using CD31 immunohistochemistry. SU11248 significantly reduced tumor weight over the control (1.22 ± 0.28 vs. 3.28 ± 0.31 g; n = 8; p < 0.01) and IMD (111 ± 10 vs. 155 ± 6 IM#/mm2; p < 0.01). RT-PCR indicated that VEGFR1 and R2 were expressed in cultured E0771 cells. VEGF (10 ng/ml) caused a 42% increase in proliferation of E0771 cells, compared to the control (p < 0.01; n = 8), and there was a significant decrease in proliferation of E0771 cells treated with VEGF plus SU11248 (10 µmol/L) vs. the control (65%, p < 0.01). VEGF caused a 2-fold increase in the proliferation of HUVEC vs. the control (p < 0.01; n = 8), but its action was completely eradicated by SU11248. Neither VEGF nor SU11248 had any effect on the proliferation of cultured HAS MC. Migration assay showed that SU11248 (10 µmol/L) significantly inhibited the migration of cultured E0771 cells. SU11248 significantly inhibited the proliferation of MCF-7 and MDAMB-231 cells in a dose-related manner. These findings support the hypothesis that the antitumor activity of SU11248 on breast cancer is possibly mediated by targeting the paracrine and autocrine effects of VEGF on breast cancer to suppress tumor angiogenesis, proliferation and migration.

Oral administration of pyrrolidine dithiocarbamate (PDTC) inhibits VEGF expression, tumor angiogenesis, and growth of breast cancer in female mice.

The progression of breast cancer is associated with oxidative stress.  However, the effects of pyrrolidine dithiocarbamate (PDTC), a known antioxidant, on the development of breast cancer are poorly understood.  The present study evaluates the effects of PDTC on tumor growth, the expression of vascular endothelial growth factor (VEGF), and angiogenesis of breast cancer in female mice.  Eight week old female mice (C57BL/6J) were given PDTC at 100 to 200 mg/kg/day for 3 weeks (n=10).  The control mice received regular drinking water only.  In the 2nd wk, 5x10^5 E0771 (mouse breast cancer) cells were injected in the pad of the fourth mammary gland of the mice.  Tumor size was monitored using dial calipers.  At the end of the experiment, the tumors were isolated and measured for tumor size, intratumoral microvessel (IM) density using CD31 immunohistochemistry staining, NFκB activation using EMSA, and VEGF protein levels using ELISA.  PDTC treatment caused a significant decrease in tumor weight compared to the control (0.64±0.22 vs. 1.43±0.31 g; n=8; P< 0.01), and a significant decrease in IM density (66.1±5.3 vs. 84.2±9.4 IM# /mm^2; P< 0.01).  There was a significant decrease in tissue protein levels of VEGF (22.6±2.1 vs. 32.4±2.6 pg/mg) and a 43% reduction NFκB activation in the breast tumors of mice treated with PDTC compared to the control group (P< 0.01).  Western blot indicated that estrogen receptor-α (ERα), VEGF receptor-1 (Flt-1), and VEGF receptor-2 (Flk-1) were expressed in E0771 cells.  VEGF receptor inhibitor SU5416 and PDTC synergistically suppressed the proliferation of E0771 cells.  PDTC also significantly inhibited the migration of cultured E0771 cells.  These results support the hypothesis that PDTC suppresses tumor angiogenesis, growth, and migration of breast cancer via inhibiting autocrine and paracrine effects of VEGF through the reduction of NFκB activation and VEGF expression.

Obesity promotes melanoma tumor growth: role of leptin.

Epidemiological studies suggest that obesity increases the risk of developing several cancers, including melanoma. Obesity increases the expression of angiogenic factors, such as leptin, that may contribute to tumor growth. However, a direct cause and effect relationship between obesity and tumor growth has not been clearly established and the role of leptin in accelerating tumor growth is unclear. Our objective in the present study was to examine the rate of melanoma tumor growth in lean and obese mice with leptin deficiency or high levels of plasma leptin. We injected 1 x 10(6) B16F10 melanoma cells subcutaneously into lean wild type (WT), obese melanocortin receptor 4 knockout (MC4R(-/-)), which have high leptin levels, obese leptin-deficient (ob(-/-)), pair fed lean ob(-/-), and lean ob(+/-) mice. Mean body weights were 29.7 +/- 0.3 g (WT), 46.3 +/- 1.9 g (MC4R(-/-)), 63.7 +/- 0.9 g (ob(-/-)), 30.5 +/- 1.0 g (pair fed ob(-/-)) and 31.6 +/- 1.7 g (ob(+/-)). Tumors were much larger in the obese leptin deficient ob(-/-) (5.1 +/- 0.9 g) and obese MC4R(-/-) (5.1 +/- 0.7 g) than in lean WT (1.9 +/- 0.3 g) and ob(+/-) (2.8 +/- 0.7 g) mice. Prevention of obesity by pair feeding ob(-/-) mice dramatically reduced tumor weight (0.95 +/- 0.2 g) to a level that was significantly lower than in WT mice of the same weight. Tumor VEGF levels were the highest in the obese mouse tumors (p < 0.05), regardless of the host leptin levels. Except for the lean ob(+/-), MC4R(-/-) and ob(-/-) melanomas had the highest VEGF receptor 1 and VEGF receptor 2 protein expression (p < 0.01 and p < 0.05), respectively. These results indicate that obesity markedly increases melanoma tumor growth rate by mechanisms that may involve upregulation of VEGF pathways. Although tumor growth does not require host leptin, melanoma tumor growth may be accelerated by leptin.

Long-term High Salt Diet Causes Hypertension and Decreases Renal Expression of Vascular Endothelial Growth Factor in Sprague-Dawley Rats.

We seek to determine: 1) whether a long-term high salt diet induces hypertension and renal injury in Sprague-Dawley (SD) rats and 2) whether the high salt diet-induced hypertension and renal injury are associated with decreased renal VEGF expression. Twelve 10-wk-old male SD rats received a high salt diet (HS, 8%) and twelve SD rats received a normal salt diet (NS, 0.5 %) for 8 weeks. Using a tail cuff, weekly monitoring showed that blood pressure increased significantly after 6, 7, & 8 wks in HS group, compared to NS group (P<0.01). At 4 wks and 8 wks of diet, mean arterial pressure (MAP) was determined in conscious rats by continuous monitoring through a catheter placed in the carotid artery. MAP was not significantly different between HS and NS group in 4 wks, but was significantly higher in HS than NS group (140+/-5.3 vs.112+/-2.2 mmHg; P<0.01) in 8 wks. Increased proteinuria and albuminuria were associated with marked renal histological abnormalities in HS group, compared to those in NS group. Northern blot and ELISA demonstrated that 8 wks of HS diet significantly decreased renal expression of VEGF mRNA and protein, compared to NS group (P<0.01). In 8 wks, total urinary excretion of sFlt-1 was significantly higher in HS than NS group (9.28+/-1.05 vs. 2.05+/-0.55 ng/day; P<0.01) whereas the plasma levels of sFlt-1 remained stable. These results suggest that a long-term HS diet induces renal injury and hypertension, which are associated with decreased renal VEGF expression in normotensive rodent animals.

Chronic alcohol consumption stimulates VEGF expression, tumor angiogenesis and progression of melanoma in mice.

The mechanisms of alcohol-induced cancer in humans are unclear. We used the immunocompetent mice implanted with B16F10 cells to evaluate the effects of physiologically relevant EtOH intake on tumor growth and angiogenesis of melanoma. Six-wk-old male mice (C57BL/6J) were given 1% EtOH in drinking water for 12-hrs during the night which was then replaced with regular water during the remaining 12-hrs each day for 4 wks (n = 10). The control mice received regular drinking water only. In the second week, all mice were inoculated subcutaneously on the right proximal dorsal with 5 x 10(5) B16F10 cells. In the end, the tumors were isolated for measuring tumor size, average microvascular density (AMVD) using CD31 immunohistochemistry, and the expression of VEGF and its receptor (Flt-1) using Northern blot, ELISA, and immunohistochemistry. EtOH intake caused a 2.16-fold increase in tumor weight over the control (4.81 +/- 0.39 vs. 2.23 +/- 0.48 g; n = 10; p = 0.003), a 2.02-fold increase in AMVD (60.63 +/- 5.56 vs. 30.01 +/- 7.41/mm(2); p = 0.0014), and a significant increase in VEGF mRNA and protein expression plus Flt-1 protein levels in melanoma compared to the control group (p < 0.01). These results suggest that progression of melanoma growth and angiogenesis may be mediated by upregulation of VEGF and Flt-1, especially under the influence of EtOH.

Exercise increases soluble vascular endothelial growth factor receptor-1 (sFlt-1) in circulation of healthy volunteers.

BACKGROUND: Physical inactivity increases the risk of cancer and atherosclerosis; the impaired regulation of angiogenesis is often associated with the development of these diseases. We hypothesize that exercise increases circulating sFlt-1, an endogenous VEGF inhibitor, which may functionally decrease plasma levels of free VEGF. MATERIAL/METHODS: 5 healthy male adults were assigned to a treadmill exercise study. The peak speed and the time spent at peak speed on the treadmill were 4.8+/-1.0 miles/h and 6.8+/-2.6 minutes, respectively. Plasma levels of sFlt-1 and VEGF were determined using ELISA (R&D Systems). RESULTS: Basal plasma levels of sFlt-1 (before exercise) were 48.8+/-9.0 pg/ml. Plasma levels of sFlt-1 increased to 72.9+/-14.6 pg/ml at 0.5 h after exercise, compared to the basal levels (49% higher, P=0.0048). The plasma levels then returned to 47.5+/-14.3 and 43.3+/-10.2 pg/ml, at 2 and 6 h after exercise, respectively. There was a significant positive correlation between % increase in plasma levels of sFlt-1 and total peak oxygen consumption during exercise (R2=0.8244; P<0.01). Basal plasma levels of unbound VEGF were 37.3+/-7.7 pg/ml, then decreased to 28.2+/-6.3, 17.5+/-2.5, and 26.6+/-6.4 pg/ml, at 0.5, 2, and 6 h, respectively, after exercise. There was a significant increase in basal plasma levels of sFlt-1 after repeated exercise for 2 weeks. CONCLUSIONS: We have demonstrated that circulating sFlt-1 is significantly increased by exercise in healthy volunteers, which is functionally associated with a transient decrease in circulating free VEGF. This data may provide new insight into the molecular links between physical inactivity and risk of cancer and atherosclerosis.

Tissue endostatin correlates inversely with capillary network in rat heart and skeletal muscles.

The role of angiostatic factors, including endostatin, in regulating physiological angiogenesis is poorly understood. We used normal adult rats under physiological resting conditions to examine the relationship between tissue endostatin, VEGF, and capillary density (CD) in the heart (high metabolic activity) versus the skeletal muscle (relatively low metabolic activity). The heart (left ventricle, LV) and skeletal muscle (anterior tibialis, AT) were dissected from 12-week-old male Sprague-Dawley rats. Transverse cryosections of LV and AT were stained with FITC-conjugated GS-I-lectin. CD was determined by analysis of randomly acquired digital images of the cryosections using Optimas software. Tissue protein levels of endostatin and VEGF were determined by ELISA assays. Tissue endostatin levels were lower in the LV and higher in the AT (135 +/- 39 vs. 663 +/- 114 pg/mg) and VEGF levels were higher in the LV and lower in the AT (41 +/- 3 vs. 27 +/- 4 pg/mg), respectively (n = 7, P < 0.01). CD in LV and AT were 3632 +/- 428 and 437 +/- 44/mm2, respectively (P < 0.01). We demonstrated that an 8.3-fold greater capillary density is related to a 4.9-fold lower level of tissue endostatin and a 1.5-fold higher level of tissue VEGF in the heart (LV) versus the skeletal muscle (AT) of normal rats under physiological resting conditions. Also, exercise training increased capillary density, decreased tissue endostatin and increased tissue VEGF in the skeletal muscle (AT). These findings suggest that tissue endostatin content correlates inversely with capillary network in the muscle tissues with different metabolic activity, and that tissue endostatin may play a very important role in the metabolic control of angiogenesis under physiological conditions.