Targeting tumor vasculature using VEGF-toxin conjugates.
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Angiogenesis Inhibitors↗
Biomedical subjects
S Ramakrishnan
Publications and source records attributed to S Ramakrishnan.
Osteoporosis--current concepts.
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Absorptiometry, Photon↗
A current perspective on clubbing and hypertrophic osteoarthropathy.
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Female↗
Insulin receptor tyrosine kinase activity in monocytes of type 2 diabetes mellitus patients receiving oral L-lysine.
The action of lysine as an antidiabetic agent was examined in human volunteers. Eight patients with type 2 DM were orally supplemented with L-lysine hydrochloride 1 g/day in two doses along with antidiabetic tablets (glyciphage or chlorformine), for a period of two months. Periodically their plasma fasting sugar and insulin receptor tyrosine kinase activity was measured in their monocytes. Eight normal healthy volunteers served as controls for comparison of receptor tyrosine kinase activity. Insulin receptor tyrosine kinase was isolated from monocytes by immunoprecipitation and the activity was determined using exogenous substrate poly glu-tyr (4:1) and radioactive ATP. Phosphorylated peptide was separated by electrophoresis and quantified using a liquid scintillation system. The enzyme activity was significantly low (22074 +/- 1728 dpm/ml immunoprecipitate) in subjects with diabetes when compared to non-diabetic control group (50,775 +/- 3597). Lysine treatment enhanced the enzyme activity by 31% in patients with diabetes and decreased their blood sugar by 27%.
Administration, Oral↗
Effect of endostatin on spontaneous tumorigenesis of mammary adenocarcinoma in a transgenic mouse model.
A transgenic mouse model was used to evaluate the effect of endostatin treatment on spontaneous tumorigenesis. In this model system, female mice develop multiple mammary adenocarcinomas and male mice develop prostate cancer. Female mice treated with mouse endostatin during a 12-15-week period showed delayed tumor development by 4-6 weeks and significantly decreased tumor burden. Furthermore, endostatin treatment reduced the number of malignant lesions per mouse. In a separate set of experiments, male mice treated with endostatin showed a survival advantage, and their life spans were prolonged by 10.5 weeks over control animals. These data demonstrate that mouse endostatin is effective in delaying spontaneous tumor development and growth.
Adenocarcinoma↗
Charge-density-wave transitions in the local-moment magnet Er5Ir4Si10
We report the observation of a new type of charge-density wave (CDW) in the large magnetic-moment rare-earth intermetallic compound, Er5Ir4Si10, which then orders magnetically at low temperatures. Single crystal x-ray diffraction shows the development of a 1D incommensurate CDW at 155 K, which then locks into a purely commensurate state below 55 K. The well-localized Er3+ moments are antiferromagnetically ordered below 2.8 K. We observe very sharp anomalies in the specific heat at 145 and 2.8 K, signifying the bulk nature of these transitions. Our data suggest the coexistence of strongly coupled CDW with local-moment antiferromagnetism in Er5Ir4Si10.
Journal Article↗
Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth.
Ovarian cancer is the leading cause of fatality among gynecological malignancies. Ovarian cancer growth is angiogenesis-dependent, and an increased production of angiogenic growth factors such as vascular endothelial growth factor is prognostically significant even during early stages of the disease. Therefore, we investigated whether antiangiogenic treatment can be used to inhibit the growth of ovarian cancer in an experimental model system. Mouse angiostatin (kringle 1-4) and endostatin were expressed in yeast. Purified angiostatin and endostatin were then used to treat established ovarian cancers in athymic mice. These studies showed that both angiostatin and endostatin inhibited tumor growth. However, angiostatin treatment was more effective in inhibiting ovarian cancer growth when compared with endostatin in parallel experiments. Residual tumors obtained from angiostatin- and endostatin-treated animals showed decreased number of blood vessels and, as a consequence, increased apoptosis of tumor cells. Subsequently, the efficacy of a combined treatment with angiostatin and endostatin was investigated. In the presence of both angiostatic proteins, endothelial cell proliferation was synergistically inhibited. Similarly, a combination regimen using equal amounts of angiostatin and endostatin showed more than additive effect in tumor growth inhibition when compared with treatment with individual angiostatic protein. These studies demonstrate synergism between two angiostatic molecules and that antiangiogenic therapy can be used to inhibit ovarian cancer growth.
Angiostatins↗
Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.
Tumor growth is angiogenesis dependent. As a consequence, strategies aimed at disrupting this mechanism are heavily investigated. Several angiogenesis assays are used to directly compare the efficacy of anti-angiogenic compounds. However, objective assessment of new vascular growth has been difficult to achieve. The aim of this study was to test and develop a computer-assisted image analysis method that would give an unbiased quantification of the microvessel density. Human tumors were grown in athymic mice and tumor biopsies were taken after a weeklong treatment with VEGF-toxin conjugate. Frozen tumor sections were prepared and stained with PE-conjugated anti-CD-31 antibodies and vessels were imaged with a fluorescence microscope. Vessel density was analyzed by quantifying PE-positive pixels per recorded field. In addition, images were further processed to investigate morphological differences by an automated binarization and skeletonization protocol. This procedure allowed the computer-assisted estimation of important angiogenic parameters such as total vessel number, length, and branch points. Based on these indices, differences in the angiogenic response between control tumors and those treated with VEGF-toxin conjugate were readily detected (P < 0.007 for all parameters). More importantly, computer-generated measurements correlated well with manual microvessel counts and showed significantly less variation. Our results suggest that computer-assisted image analysis represents a rapid, objective, and alternative method for the quantitative assessment of tumor angiogenesis and vessel architecture.
Adenocarcinoma↗
Homocystinuria with congenital/developmental cataract.
The aim of the study is to screen patients for homocystinuria with and without cataract and analyse for homocystine and methionine. Fifty-eight samples from 29 patients, i.e., plasma and urine collected after overnight fasting were analysed by the screening test for homocystine, and paper chromatography for homocystine and methionine. Out of 29 homocystinuric patients, 24 had cataract. Only one had appreciable amounts of methionine in his serum. He also had mental retardation as expected and belongs to Type I. The other types did not have methionine but had only homocystine. There was no mental retardation or ectopia lentis. So they belonged to Types II, III or IV. As there is excess methionine in Type I, with low cystine, cataract may be due to deficiency of cysteine and reduced glutathione and might be averted by suitable therapy, i.e., high cystine-low methionine diet with B6. In other types with low methionine, cataract may be due to decreased availability of amino acids for the synthesis of lens proteins; the treatment of choice should be B12, and folate with methionine.
Adult↗
Inhibition of angiogenesis and tumour growth by VEGF121-toxin conjugate: differential effect on proliferating endothelial cells.
Vascular endothelial growth factor (VEGF) plays an important role in tumour angiogenesis. VEGF binds to tyrosine kinase receptors, which are expressed almost exclusively on tumour endothelium. Therefore, VEGF can be used to target toxin molecules to tumour vessels for anti-angiogenic therapy. However, recent evidence suggests that VEGF can also bind in an isoform-specific fashion to a newly identified neuropilin-1 (NP-1) receptor. NP-1 is widely expressed in normal tissue and presents a potential target for unwanted toxicity. As a consequence, we investigated whether the VEGF121 isoform, which lacks the NP-1 binding domain, could be used to target toxin polypeptides to tumour vasculature. Treatment of endothelial cells with a VEGF121-diphtheria toxin (DT385) conjugate selectively inhibited proliferating endothelial cells, whereas confluent cultures were completely resistant to the construct. In addition, VEGF121-DT385 conjugate treatment completely prevented tumour cell induced angiogenesis in vivo. Most importantly, the conjugate inhibited tumour growth in athymic mice and induced tumour-specific vascular damage. There was also no apparent toxicity associated with the treatment. Our results suggest that proliferating endothelial cells are highly sensitive to VEGF121-toxin conjugates and that the binding to NP-1 receptors is not necessary for efficient inhibition of tumour growth.
Allantois↗
Circulating lipoprotein profiles are modulated differently by lipoprotein lipase in obese humans.
BACKGROUND: Several genetic analyses have suggested that lipoprotein lipase (LpL) genotypes causing decreased LpL activity correlate with increased triglyceride concentrations and risk for coronary artery disease. In contrast, in some other studies LpL activity was positively correlated with plasma low-density lipoprotein (LDL) cholesterol concentrations. OBJECTIVE: To assess whether these different associations represent physiologic differences in lipoprotein metabolism. METHODS: We correlated postheparin lipase activities, postprandial lipemia, and fasting lipoprotein concentrations in obese (BMI > or = 30 kg/m2, n = 26) and non-obese (BMI < or = 30 kg/m2, n = 57) individuals. LpL was measured using specific inhibitory antibodies. RESULTS: Surprisingly, LpL activity was significantly correlated with triglyceride area under the curve after a fat load in the non-obese, but not the entire group. Moreover, in non-obese individuals, LpL activity correlated directly (r = 0.40) and hepatic lipase activity correlated inversely (r = -0.32) with high-density lipoprotein (HDL) cholesterol concentrations. These relationships were not found in the obese group, in whom LpL correlated with LDL cholesterol concentrations (r = 0.54). CONCLUSIONS: We conclude that postheparin LpL activity relates to different lipoproteins in obese and non-obese individuals. In obesity, greater LpL activity may enhance conversion of very-low-density lipoprotein cholesterol to LDL cholesterol, whereas in non-obese individuals the correlation is with HDL cholesterol. Whether this is due to differences in the source of LpL (muscle or fat), or to other associated alterations in lipoprotein metabolism is unknown. These results may explain the non-uniformity of correlations between LpL and atherogenic lipoproteins in different populations.
Adult↗
Erythrocyte deformability in Waldenström's macroglobulinemia.
The deformability of erythrocytes in Waldenström's Macroglobulinemia (WM) patients are analysed by shear stress diffractometer in vitro. Erythrocytes obtained from blood samples of WM patients are subjected to well defined shear forces, varying from 0.3 to 60 Pa, and their corresponding elongation indexes generated by diffraction pattern of a laser beam are recorded. For comparison, equal number of samples from normal adult volunteers is also studied. The results show a shear dependent decrease in deformability index in WM patient samples. The decrease in deformability index of WM patients is significant at high shear forces (>12 Pa) when compared to those of normal samples whereas it is not significant at low shear forces. As the deformability of erythrocytes together with elevated plasma viscosity and enhanced erythrocyte aggregation may cause potential flow stagnation at microvasculature, these studies are clinically relevant.
Erythrocyte Deformability↗
Possible role of polyamines in gyrate atrophy.
PURPOSE: Gyrate atrophy (GA) is marked by hyperornithinemia and lowered ornithine amino transferase (OAT). However there are patients of GA without hyperornithinemia and those with hyperornithinemia without GA. Some cases of GA have been reported to have low lysine. The purpose of the study was to determine if polyamines, the metabolites of ornithine, and lysine have any diagnostic role in GA. METHODS: Ornithine in plasma was estimated by two-dimensional paper chromatography, with elution of the coloured spot, and the absorbance measured using a spectrophotometer at 560 nm. OAT assay in lymphocytes was done spectrophotometrically using ornithine as substrate. Blood and urinary polyamines were extracted with n-butanol, benzoylated and analysed with HPLC; putrescine, spermine, spermidine, and cadaverine were assayed individually at 254 nm with the UV detector using ODS, G18 column with 63% methanol as solvent. RESULTS: Of the 7 patients investigated, 6 had features typical of GA. One was diagnosed to have atypical retinitis pigmentosa (case 3). The first five cases had elevated ornithine and diminished OAT, but cases 6 and 7 had near-normal ornithine and case 7 had near-normal OAT. However, all 7 patients had increased levels of total polyamines in urine compared to normals. Five had increased putrescine and three had increased spermine. All the 7 had decreased cadaverine in urine. Thus, though there were inconsistencies with ornithine and OAT, all the 7 patients had elevated polyamines from ornithine and decreased cadaverine. CONCLUSION: In addition to estimating ornithine and OAT in GA, it is suggested that urinary polyamines may be analysed as the latter appears to correlate better with the clinical condition and help in the diagnosis to a greater extent. Moreover, while ornithine is an innocuous amino acid, polyamines are known to damage DNA and proteins.
Adolescent↗
Intestinal cholesterol synthesis and mobilisation to blood in sucrose-feeding and alcohol-intake.
Small intestines synthesize cholesterol to a greater extent than liver. Between starch-fed and sucrose-fed rats, using (14C) glucose, it was found that the synthesis of cholesterol by the jejunum of small intestines was greater in the sucrose-fed group than starch-fed group. By a novel experimental technique and using (14C) glucose, it was found that the contribution towards buffer representing lymph was greater in the sucrose-fed group (13.3%) than the controls (11%). Hypercholesterolemia on sucrose feeding may be at least partly due to contribution by the small intestines. Regarding alcohol, using (14C) glucose it was found that total synthesis of cholesterol by the small intestines was decreased in alcohol-fed rats. There was no difference in the cholesterol retained by the intestinal tissue between the controls and alcohol-fed animals while, the secretion towards buffer (lymph) was 9% as against 11. This indicates that there is contribution of cholesterol to blood from small intestines in alcohol-intake also but due to overall decrease in the intestinal synthesis of cholesterol, contribution of intestines to hypercholesterolemia may not be substantial as in the case of sucrose feeding. This is because in sucrose-feeding there is increased cholesterol synthesis.
Animals↗
Sickle cell anemia as a possible state of enhanced anti-apoptotic tone: survival effect of vascular endothelial growth factor on circulating and unanchored endothelial cells.
The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% +/- 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% +/- 15% (P <.001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P =.001) higher in plasma of sickle donors (120.1 +/- 81.4 pg/mL) than that of normal donors (37.6 +/- 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r =. 612, P =.001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of alphavbeta3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.
Adult↗
VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling.
Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes endothelial cell survival and angiogenesis. We recently showed that VEGF can support the growth of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells in serum-free medium. Reasoning that VEGF might be modulating apoptotic signal transduction pathways, we examined mechanisms involved in the anti-apoptotic effect of VEGF on starvation- and ceramide-induced apoptosis in HDMEC. We observed that VEGF ameliorated the time-dependent increase in apoptosis, as demonstrated by morphologic observations, TUNEL assay, and DNA fragmentation. On the other hand, basic fibroblast growth factor only partially prevented apoptosis in serum-starved HDMEC; platelet-derived growth factor-BB was completely ineffective. VEGF activated the phosphorylation of extracellular signal regulated kinase (ERK)1 (p44 mitogen-activated protein kinase; MAPK) and ERK2 (p42 MAPK) in a time- and concentration-dependent manner. Both the VEGF-induced activation and its anti-apoptotic effect were prevented by the specific MAPK/ERK inhibitor PD98059. The presence of VEGF also inhibited the sustained activation of stress-activated protein kinase/c-jun-NH2-kinase (SAPK/JNK) caused by serum starvation and ceramide treatment. Activation of the MAPK pathway together with inhibition of SAPK/JNK activity by VEGF appears to be a key event in determining whether an endothelial cell survives or undergoes programmed cell death.
Apoptosis↗
Binding and displacement of vascular endothelial growth factor (VEGF) by thrombospondin: effect on human microvascular endothelial cell proliferation and angiogenesis.
Vascular endothelial growth factor (VEGF) is a specific angiogenic factor, and thrombospondin (TSP), is a potent inhibitor of angiogenesis. To better understand the role of TSP as an anti-angiogenic agent, we have identified its specific domains that participate in its anti-angiogenic activity and examined the mechanism of its inhibitory effect on VEGF(165) induced angiogenesis. Exogenously added TSP inhibited VEGF(165) induced angiogenesis (proliferation and tube formation of human dermal microvascular endothelial cells [HDMEC] and neovascular outgrowth from human arterial rings). Although both VEGF(165) and TSP are heparin binding proteins, TSP had a higher affinity for (125)I-heparin than VEGF(165) (K(d1) 4 nM and K(d2) 14 nM for TSP; K(d) 91 nM for VEGF(165)). TSP displaced 36% of (125)I-VEGF(165) from HDMEC and this was comparable to the 27% reduction in (125)I-VEGF(165) binding to HDMEC upon cleavage of cell surface heparan sulfate (HS). About 35% of the mitogenic activity of VEGF(165) was attributable to its heparin binding region. These results indicate that a proportion of the mitogenic activity of VEGF(165) is inhibited by TSP via competition for cell surface HS. Further, (125)I-VEGF(165) bound directly to TSP in a saturable, concentration dependent manner, and heparin modulated this binding. The mAbs to the heparin binding domain to the type 1 and type 3 repeats of TSP inhibited the binding of VEGF(165) to TSP, and also blocked the inhibitory effect of TSP on VEGF(165) induced HDMEC proliferation. We conclude that (i) the anti-angiogenic activity of TSP is localized in its heparin binding domain and type 1 and type 3 repeats (ii) TSP inhibits angiogenesis by at least two separate mechanisms, (a) displacement of VEGF(165) from endothelial cell HS and (b) direct binding to VEGF(165).
Journal Article↗
Eales' disease: increased oxidation and peroxidation products of membrane constituents chiefly lipids and decreased antioxidant enzymes and reduced glutathione in vitreous.
PURPOSE: To measure the levels of oxidation and peroxidation products of membrane constituents chiefly lipids produced by oxygen and lipid free radicals as thiobarbituric acid reacting substances (TBARS), reduced glutathione (GSH) and the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GP) in vitreous samples from patients with Eales' disease and estimate GSH, SOD, GP in the erythrocytes of patients with Eales' disease with active perivasculitis and healed perivasculitis. METHODS: Vitreous samples on vitrectomy from 11 patients with Eales' disease and 11 patients with diabetic vitreous hemorrhage were used for comparison. For study on erythrocytes, 30 patients of Eales' disease at the active vasculitis stage (group-I), 33 of healed vasculitis stage (group-II) and 25 controls (group-III) were used. The male female ratio was kept the same among the patients and the controls. Oxidation and peroxidation products of membrane constituents chiefly lipids were estimated as TBARS, SOD by its activity to inhibit auto-oxidation of epinephrine, GP by estimating the reduction in the level of GSH and GSH by its colour reaction with 5,5'-dithio bis (2-nitro) benzoic acid (DTNB). RESULTS: TBARS were increased 6 fold in the vitreous of Eales' patients compared to the samples from those with diabetic vitreous hemorrhage while there was a reduction of 95.9% of SOD and 84.2% of GSH. Activity of SOD, GP and the levels of GSH in the erythrocytes were reduced (%) by 81.6, 65 and 56.5 respectively in group I and 22,46.4 and 29.2% respectively in group II. The values are statistically significant. CONCLUSIONS: Increased levels of TBARS and decreased levels of SOD and GSH in the vitreous could explain inflammation, retinal damage and neovascularization in patients with Eales' disease. Decrease of SOD, GP and GSH is found in erythrocytes both in the active perivasculitis stage and the healed perivasculitis stage. Treatment of Eales' disease with antioxidants vitamin E, C as also vitamin A may have a beneficial effect.
Adult↗