Theranostic Gold Nanomicelles made from Biocompatible Comb-like Polymers for Thermochemotherapy and Multifunctional Imaging with Rapid Clearance.

A new generation of photothermal theranostic agents based on assembling 6 nm gold nanoparticles (AuNPs) is developed by using a novel comb-like amphipathic polymer as the template. The small AuNPs are assembled into DOX@gold nanomicelles, which show strong absorbance in the near-infrared region, for multimodal bioimaging and highly effective in vivo chemotherapy and photothermal therapy.

Diallyl disulfide and diallyl trisulfide protect endothelial nitric oxide synthase against damage by oxidized low-density lipoprotein.

Garlic is viewed as an effective health food against atherosclerosis. In this study, we examined whether diallyl disulfide (DADS) and diallyl trisulfide (DATS) protect endothelial nitric oxide synthase (eNOS) activation against oxidized LDL (ox-LDL) insult and through what mechanism. We found that DADS and DATS reversed the suppression of eNOS Ser1177 phosphorylation by ox-LDL, and wortmannin abolished the reversal by DADS and DATS. Similarly, the inhibition of cellular cGMP and nitric oxide production by ox-LDL was reversed by DADS and DATS (p<0.05). This increase in nitric oxide bioavailability by the allyl sulfides was attenuated by wortmannin. Immunoprecipitation assay revealed that DADS and DATS preserved the interaction of eNOS with caveolin-1 in the membrane. In addition, DADS and DATS suppressed the reduction of the cellular eNOS protein content by ox-LDL. When cycloheximide was added to block protein synthesis, DADS and DATS suppressed eNOS protein degradation similarly to that noted by MG132. Ox-LDL increased chymotrypsin-like proteasome activity, and this increase was inhibited by the allyl sulfides and MG132 (p<0.05). These results suggest that DADS and DATS protect eNOS activity against ox-LDL insult. This protection can be attributed partly to their mediation of phosphatidylinositol 3-kinase/protein kinase B signaling and prevention of eNOS degradation.

Heterogeneity of phospholipase D activation by angiotensin II, lysophosphatidylcholine, and insulin in human endothelial cells.

Human endothelial cells (ECs) are heterogeneous, although little is known regarding regional variations in their regulation of vascular tone. This study compares activation of the key enzyme phospholipase D (PLD) by the vasoconstrictors angiotensin II (AII) and lysophosphatidylcholine (lysoPC), and the vasodilator insulin, in primary human microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs). PLD activity was measured by [(3)H]phosphatidylethanol production in cells labeled with [(3)H]myristic acid. AII maximally activated PLD in both cell types at 1 nmol/L. AII also significantly activated PLD at 100 pmol/L in HUVECS but not in HMVECs. LysoPC dose dependently activated PLD in both cell types, although HUVECs were more sensitive to the agonist; being significantly activated by 10 micromol/L lysoPC and displaying an approximately sevenfold greater PLD activity with 20 micromol/L lysoPC compared to HMVECs. Insulin significantly increased PLD activity in both cell types with maximum activation at 1 nmol/L. Again differential sensitivity was observed; 10 nmol/L insulin significantly stimulated PLD in HUVECs but not HMVECs. Differential sensitivity of PLD activation in human endothelial cells from different vascular beds in response to vasoactive agents was observed, with the HUVECs displaying greater sensitivity to vasoconstricting agents than HMVECs.

Effects of four medicinal herbs on human vascular endothelial cells in culture.

BACKGROUND: Danshen (DS, Salvia miltiorrhiza), Shanchi (SQ, Panax notoginseng), Shanzai (SZ, Hawthorn) and Heshouwu (HSW, Polygonum multiflorum Thunb) are four medicinal herbs commonly used in traditional Chinese medicine and previously shown to have activity that may contribute to cardiovascular protection. This study aims to investigate effects of these herbs on vascular endothelial cells with respect to cell viability and expression of cellular adhesion molecules under inflammatory conditions. METHODS: Herbal extracts were prepared by an established industrial manufacturing process. Human umbilical vein endothelial cells (HUVEC) were incubated with the herbal extract under normal or serum-free culture and tumor necrosis factor (TNF) alpha stimulation. Cell apoptosis, apoptosis-associated gene expression, expression of cellular adhesion molecules, DNA synthesis, and growth were assessed via morphological examination, Annexin-V staining, Western blotting analysis, Flow-Cytometry, [(3)H]-thymidine incorporation assay, and cell number study. RESULTS: SZ and HSW significantly inhibited apoptosis in HUVEC undergoing serum deprivation and TNFalpha stimulation, accompanied by down-regulation of caspase-3 gene expression. DS and SQ significantly attenuated TNFalpha-induced expression of adhesion molecule VCAM-1 and also ICAM-1 by DS in the cells. All four herbs at therapeutic concentrations (100 microg/mL) inhibited DNA synthesis (10-42% decrease in [(3)H]-thymidine incorporation rates) and growth (5-10% decrease in cell numbers) in HUVEC under normal cultures. CONCLUSION: DS, SQ, SZ and HSW are physiologically active on human vascular endothelial cells. The actions by HSW and SZ to reduce apoptosis and DS and SQ to inhibit adhesion molecule expression may help protect endothelial function and inhibit atherogenesis, while their actions to inhibit DNA synthesis and cell growth may weaken the ability of endothelial repair. Further studies are needed to identify the chemical compounds responsible to these physiological effects by these herbs.

The effect of trophic factor supplementation on cold ischemia-induced early apoptotic changes.

We have previously shown that trophic factor supplementation (TFS) of University of Wisconsin (UW) solution enhanced kidney viability after cold storage. Here, we use an in vitro model to study the effect of TFS on early apoptotic changes after cold ischemic storage. Mitochondrial membrane potential was determined by fluorescence intensity in primary canine kidney tubule cells, Madin-Darby canine kidney cells, and human umbilical vein endothelial cells. In addition, caspase 3 enzyme activity assay and immunofluorescence staining were performed to evaluate apoptosis. There was a 15% increase in mitochondrial membrane potential in human umbilical vein endothelial cells stored in trophic factor supplemented University of Wisconsin solution after four-hour rewarming (P<0.05). TFS suppressed caspase 3 enzyme activity and activation in human umbilical vein endothelial cells. We confirmed that the presence of TFS in UW solution has a beneficial effect by protecting mitochondrial function and reducing early apoptotic changes in vascular endothelial cells.

Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells.

Salviae miltiorrhizae (SM), a clinical, commonly used herb, can activate blood circulation and resolve stasis. We have investigated the effects of salvianolic acid B (Sal B), a pure compound extracted from the dried SM roots, on fibrinolytic (tissue-type plasminogen activator and plasminogen activator inhibitor, t-PA and PAI) and anticoagulant (thrombomodulin,TM) properties of cultured human umbilical vein endothelial cells (HUVECs). When HUVECs were treated with Sal B, a dose- (0.0125-0.5 mg/ml) and a time-dependent decrease in PAI activity were observed. PAI type 1 (PAI-1) antigen and PAI-1 mRNA expression significantly decreased compared to control values in the conditioned media of HUVECs pretreated with Sal B for 12 h. Moreover, TM activity reached a maximum stimulation of 1.25-fold over control levels in the pretreatment of Sal B for 12 h and t-PA and TM specific mRNA expression also increased (1.7- and 1.8-fold, respectively). In conclusion, Sal B increased the fibrinolytic and anticoagulant potential of cultured HUVECs by up-regulating the expression of t-PA and TM and by down-regulating the expression of PAI-1. These data suggest that Sal B is clinically effective because of its ability to change the gene expression profile of endothelial cells thereby preventing vascular events.

Neurologic condition of healthy term infants at 18 months: positive association with venous umbilical DHA status and negative association with umbilical trans-fatty acids.

Prenatal long-chain polyunsaturated fatty acids (LCPUFAs) and trans-fatty acids may affect neurodevelopment. In healthy term children, we determined relationships between relative fatty acid contents of umbilical arteries and veins and neurodevelopment at 18 mo. The study comprised a mixed group of 317 breast-fed, formula-fed, and LCPUFA formula-fed children. Study endpoints were the Hempel neurologic examination resulting in a neurologic classification and neurologic optimality score (NOS), and the Bayley Psychomotor Developmental Index (PDI) and Mental Developmental Index (MDI). Fifteen children showed minor neurologic dysfunction (MND). The umbilical vein trans, trans-18:2n-6 content was higher in children with MND than in the normal group. The NOS was significantly reduced in infants with an umbilical vein docosahexaenoic acid (DHA) content within the lowest quartile. Umbilical vein arachidonic acid (AA) was related to NOS in univariate statistics but not in multivariate analyses. The sum of trans-fatty acids and that of C18 trans-fatty acids showed a negative association with NOS in both univariate and multivariate analyses. No associations were found between AA, DHA and total trans-fatty acids with PDI or MDI. In conclusion, neonates with a relatively low DHA status and those with high trans-fatty acid levels have a less favorable neurologic condition at 18 mo.

Double-stranded RNA induces the synthesis of retinoic acid-inducible gene-I in vascular endothelial cells.

Viral infection induces various responses in vascular endothelial cells. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), and treatment of cells with poly IC mimics the viral infection to the cells. Retinoic acid-inducible gene-I (RIG-I) is a protein belonging to the DExH-box family and designated as a putative RNA helicase. RIG-I is considered to play a role in antiviral responses through the regulation of gene expressions. In the present study, the authors treated human umbilical vein endothelial cells (HUVECs) with poly IC and found that poly IC induced the expression of RIG-I. The poly IC-induced RIG-I expression was inhibited by the preincubation of the cells with 2-aminopurine, an inhibitor of dsRNA-dependent protein kinase (PKR). Immunohistochemical examination revealed high levels of RIG-I immunoreactivity in vascular endothelial cells in the thalamus from rats inoculated with hantavirus. Induction of RIG-I by poly IC may be involved in the antiviral responses in endothelial cells.

Enhanced gene transfer activity of peptide-targeted gene-delivery vectors.

We have evaluated the capacity of the cell-binding heptapeptide SIGYPLP to enhance transgene expression using non-viral and viral gene delivery vectors. Targeted polyplex based vectors showed good levels of DNA uptake in freshly isolated human umbilical vein endothelial cells (HUVECs) compared to untargeted controls, whilst displaying only modest increases in reporter gene activity. The targeted polyplexes showed reduced levels of DNA uptake in cells of a none endothelial origin although they mediated higher levels of transgene expression. The enhanced efficiency of transgene expression may relate to the more rapid rate of cell division. However, since in vivo application of polyplexes is compromised by instability to serum proteins, serum-resistant polyplexes (surface modified with multivalent reactive hydrophilic polymers based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA)) were also evaluated for their ability to mediate transgene expression. Surface modification of polyplexes with pHPMA ablates non-specific cell entry, reducing levels of transgene expression, whilst the incorporation of the SIGYPLP peptide into the hydrophilic polymer resulted in restored transgene expression in all formulations tested. The technology of surface modification using pHPMA can also be applied in the context of viruses, masking receptor-binding epitopes and enabling the linkage of novel cell targeting ligands, enabling construction of a virus with receptor-specific infectivity. Retargeting of adenovirus based vectors using the same polymer-peptide construct enhanced levels of transgene expression in HUVECs to greater than 15 times that observed using parental (unmodified) virus, whilst restoring levels of transgene expression in non-endothelial cell lines tested. The use of constructs based on conjugates between hydrophilic polymers and small receptor-binding oligopeptides as agents for retargeting viral or non-viral vectors to cellular receptors represents a simple alternative to the use of antibodies as targeting ligands for cell specific gene delivery.

Mesenchymal progenitor cells in the human umbilical cord.

Mesenchymal progenitor or stem cells (MPCs) isolated from fetal blood, liver, and bone marrow are a population of multipotential cells that can proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat, and stroma. The objective of this study was to isolate and characterize MPCs in the human umbilical cord. The suspensions of endothelial and subendothelial cells in cord vein were collected and cultured in M199 supplemented with 10% fetal bovine serum (FBS). Of 50 umbilical cord samples, 3 had numerous fibroblastoid cells morphologically distinguishable from endothelial cells. Fibroblastic cells displayed lack of expression of vWF, Flk-1, and PECAM-1, indicating the endothelial cell-specific marker. To investigate the differentiation potentials, the cells were cultured in adipogenic or osteogenic medium for 2 weeks. Fibroblast-like cells treated with adipogenic supplementation showed Oil red O-positive staining and expressed adipsin, FABP4, LPL, and PPARgamma2 genes by reverse transcriptase polymerase chain reaction (RT-PCR). In osteogenic differentiation, alkaline phosphatase activity and calcium accumulation were detected. RT-PCR studies determined that Cx43, osteopontin, and Runx2 genes were expressed in the osteogenic cultures. Among three cell lines cultured continuously for passage 10, two had normal karyotypes; however, one retained a karyotype of mos 46,XY[19]/47,XY,+mar[3]. These observations suggest that MPCs are present in human umbilical cord and possess several typical traits of MPCs.

Nerve growth factor supplementation reverses the impairment, induced by Type 1 diabetes, of hindlimb post-ischaemic recovery in mice.

AIMS/HYPOTHESIS: Type 1 diabetes increases the risk of peripheral ischaemia and impairs recovery once ischaemia occurs, probably because the healing process is hampered by diabetes-induced endothelial dysfunction. In normoglycaemic mice subjected to limb ischaemia, blockade of nerve growth factor (NGF) compromises reparative angiogenesis. In the present study, we evaluated if expressional alterations of endogenous NGF system components are associated with diabetes-related impairment in neovascularisation. In addition, we tested whether the correction of NGF liabilities benefits post-ischaemic healing of Type 1 diabetic animals. METHODS: Unilateral hindlimb ischaemia was produced in streptozotocin-induced Type 1 diabetic mice. Purified murine NGF (20 microg daily for 14 days) or PBS were injected into ischaemic adductors. Non-diabetic mice given PBS served as controls. Hindlimb blood flow was analysed sequentially for up to 14 days. At necroscopy, adductors were removed for quantification of microvessel density, endothelial cell apoptosis and NGF receptor expression. NGF content was determined by ELISA three days after ischaemia. In vitro, we tested whether NGF protects endothelial cells from apoptosis induced by high glucose and whether vascular endothelial growth factor-A (VEGF-A) is involved in this beneficial effect. RESULTS: Muscles removed from Type 1 diabetic mice showed reduced NGF content and up-regulation of the NGF p75 receptor. NGF supplementation promoted capillarisation and arteriogenesis, reduced apoptosis, and accelerated blood flow recovery. NGF stimulated VEGF-A production by human endothelial cells incubated in high-glucose medium and conferred resistance against high-glucose-induced apoptosis via a VEGF-A-mediated mechanism. CONCLUSIONS/INTERPRETATION: NGF protects endothelial cells from apoptosis induced by Type 1 diabetes and facilitates reparative neovascularisation. The findings may open up new therapeutic options for the treatment of diabetic complications.

Class-specific pro-apoptotic effect of statins on human vascular endothelial cells.

Neonangiogenesis represents an important step in tumor development and propagation. Statins may have anticancerogenic potential by blocking vascular endothelial cell growth. The antiproliferative effect of four statins on human endothelial cells was compared, concomitantly delineating a possible pro-apoptotic process. All four statins tested, i. e. atorvastatin, fluvastatin, lovastatin, and simvastatin inhibited cell proliferation. Nearly complete blocking of cell proliferation was achieved at a concentration of 10 microM. We were able to demonstrate that the antiproliferative effect of the statins is not due to cytotoxicity but rather to an apoptotic effect as demonstrated by comparison of cytotoxicity assay and apoptosis assay. The apoptotic mechanism seems to involve caspases, since the statins significantly enhanced caspase activity at dosages of 10 and 20 microM. Further experiments revealed a downregulation of the pro-apoptotic protein Bcl-2. Our data indicate that statins may class-specific inhibit angiogenesis at high dosages which can contribute to prevention of tumor development and progression.

Further pharmacological evidence of nuclear factor-kappa B pathway involvement in bradykinin B1 receptor-sensitized responses in human umbilical vein.

Bradykinin (BK) B(1) receptors are thought to exert a pivotal role in maintaining and modulating inflammatory processes. They are not normally present under physiological situations but are induced under physiopathological conditions. In isolated human umbilical vein (HUV), a spontaneous BK B(1) receptor up-regulation and sensitization process has been demonstrated. Based on pyrrolidine-dithiocarbamate inhibition, it has been proposed that this phenomenon is dependent on nuclear factor-kappaB (NF-kappaB) activation. The aim of this study was to further evaluate the NF-kappaB pathway involvement on BK B(1) receptor sensitization in isolated HUV, using several pharmacological tools. In 5-h incubated rings, either the I-kappaB kinase inhibitor 3-(4-methylphenylsulfonyl)-2-propenenitrile (Bay 11-7082) or the proteasome activity inhibitor Z-Leu-Leu-Leu-CHO (MG-132) inhibited the development of the BK B(1) receptor-sensitized contractile responses. Furthermore, pro-inflammatory cytokine interleukin-6 (IL-6) produced a leftward shift of the concentration-response curve to the BK B(1) receptor agonist, whereas anti-inflammatory cytokines interleukin-4 (IL-4) and tumor growth factor-beta1 (TGF-beta1) produced a rightward shift of the responses to des-Arg(9)-BK in our preparations. Taken together, these results point to NF-kappaB as a key intermediary in the activation of the expression of BK B(1) receptor-sensitized responses in HUV and support the role of inflammatory mediators in the modulation of this process.

Novel antiallergic and antiinflammatory agents. Part II: Synthesis and pharmacology of TYB-2285 and its related compounds.

A series of m-bis(glycoloylamino)benzene derivatives was synthesized by treatment of the corresponding m-diaminobenzene derivatives with glycoloyl chloride derivatives in pyridine. Hydrolysis of acetyl compounds gave hydroxy derivatives, from which other acyl derivatives could be synthesized. These compounds were tested in the rat PCA (passive cutaneous anaphylaxis) assay by oral administration. Benzonitrile derivatives (4c, 5c, 6c, 4h, 5h) exhibited notable inhibition in this assay. Compounds 5c and 6c also showed remarkable inhibition of eosinophil adhesion to TNF- (tumor necrosis factor) alpha-treated HUVEC (human umbilical vein endothelial cells) in the range of 10(-8)-10(-5) M. Compound 5c is now under investigation in Japan as TYB-2285 (Figure 1) for asthma and atopic dermatitis in phase II clinical studies.

Effects of intrauterine nutritional supplementation on fetal growth retardation.

The effects of fetal nutritional supplementation on experimentally induced growth retardation were investigated in third-trimester sheep. The control group, C, was fed ad libitum (n = 8). Group R received a protein- and calorie-restricted diet over the last 3 weeks of gestation (n = 8). Group RS was given the same restricted diet as group R, and fetal, intragastric supplements of amino acids and glucose (n = 7). Over the experimental period, maternal weight increased 4.1 +/- 0.6 kg in C but was stable in R and RS (p less than 0.01). R and RS ewes and fetuses showed biochemical evidence of malnutrition but the changes were less severe in the RS group (p less than 0.05). Nutrient levels did not change in group C ewes and fetuses. At delivery, R lambs were smaller in weight and length than RS or C (p less than 0.05); gestational ages were the same. Weights were: R, 2,702 +/- 182 g; RS, 3,235 +/- 121 g; and C, 3,290 +/- 297 g. R also had lower fetal to maternal weight ratios and higher brain to body weight ratios than RS or C (p less than or equal to 0.05). Placental size was reduced 28% in R compared to C (p less than 0.05) and was intermediate in RS. The disparity in fetal size was explained by differences in nutrient acquisition between groups. Over the experimental period, caloric intake rose 24% in C fetuses; near delivery, they had the highest umbilical uptakes of carbohydrate and alpha-amino nitrogen. After maternal diet was limited, R and RS fetuses had dramatic decreases in umbilical uptake of alpha-amino nitrogen and static umbilical uptakes of carbohydrate. Caloric intake fell 42% in R. RS fetuses, however, were receiving sufficient gastric supplementation so that umbilical + gastrointestinal carbohydrate uptake equalled that of controls, and entry of alpha-amino nitrogen and calories was intermediate, between those of R and C. Fetal oxygen consumption reflected the differences in nutrient uptake, being greatest in C, lowest in R and intermediate in RS. Fetal supplementation lessened the drain on maternal nutrients and promoted more normal fetal growth and metabolism.