Novel Angiogenic Compounds for Targeted Drug Delivery

2004 ◽  
Vol 845 ◽  
Author(s):  
Kristen A. Wieghaus ◽  
Scott M. Capitosti ◽  
Milton L. Brown ◽  
Edward A. Botchwey
Keyword(s):  
Cell Death ◽  
Wound Repair ◽  
Culture Conditions ◽  
Hypoxic Cell ◽  
Growth Factor Delivery ◽  
Microvascular Networks ◽  
Highly Sensitive ◽  
Endothelial Cell Death ◽  
Graft Reconstruction

ABSTRACTInduction of angiogenesis is necessary for the success of engineered implantable tissues in order to meet oxygen and nutrient requirements of cells during tissue repair. Insufficient vascularization in bone graft reconstruction may impede healing and initiate hypoxic cell death at the interior of the implant. As a result, endogenous growth factors have been studied to enhance angiogenesis during wound repair. However, these peptide-based molecules are highly sensitive to processing that occurs during scaffold biomaterial fabrication and treatment for tissue engineering purposes. We report here the development of new small molecule regulators of angiogenesis that may circumvent the impediments associated with protein-based growth factor delivery. In this study, we report the design and evaluation of SC–3–143 as a regulator of endothelial function. We show that the compound significantly increases the formation of microvascular networks in vitro, and selectively enhances endothelial survivability by reducing endothelial cell death under serum deprived culture conditions.

Abstract 4469: Carbamoylating activity associated with the antitumor prodrug Laromustine inhibits angiogenesis in vitro by inducing ASK1-dependent endothelial cell death

2011 ◽  
Author(s):  
Roxanne Ghazvinian ◽  
V. Alexandra Praggastis ◽  
Xianghong Li ◽  
Wei Zhang ◽  
Edmund J. Klinkerch ◽  
...  
Keyword(s):  
Cell Death ◽  
Endothelial Cell ◽  
Endothelial Cell Death

Vascular Endothelium Is Severely Perturbed and Undergoes Apoptosis in Experimental Heatstroke in Primates.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3972-3972
Author(s):  
George T. Roberts ◽  
Muhammad A. Chishti ◽  
Fallah H. Al-Mohanna ◽  
Raafat M. El-Sayed ◽  
Abderezak Bouchama
Keyword(s):  
Cell Death ◽  
Endothelial Cell ◽  
Vascular Endothelium ◽  
Apoptotic Cell ◽  
Chromatin Condensation ◽  
Apoptotic Cell Death ◽  
Control Group ◽  
Tissue Sections ◽  
Endothelial Cell Death

Abstract Introduction: Ultrastructural evidence of endothelial cell (EC) injury has been associated with diffuse microvascular thrombosis in human heatstroke (HS). In vitro studies have also shown that heat stress accelerates apoptotic cell death. Using a recently described baboon model of heatstroke, we sought to examine pathological changes in the vascular endothelium and whether apoptosis is a mechanism of endothelial cell death. Hypothesis: Major structural vascular endothelium alterations occur in HS and apoptosis is a mechanism of endothelial cell death in HS. Methods: Anesthetized baboons (Papio hamadyras) were heat-stressed in a neonatal incubator maintained at 44 1.5 °C, until rectal temperature attained 42.5°C (moderate heatstroke; n =4) or systolic blood pressure fell to < 90 mm Hg (severe heatstroke n =4). Animals were resuscitated with normal saline and allowed to cool at room temperature. Four sham-heated animals served as control group. Spleen, liver, heart, kidney, gut, lung and adrenal tissue were obtained either by immediate autopsy in non-survivors or after euthanasia at 72-h for survivors. Vascular endothelium ultrastructure was evaluated by transmission electron microscopy (TEM) of ultra-thin tissue sections. Biological activity of EC was determined by light microscopy (LM) using a polyclonal antibody targeting von Willebrand Factor (vWF). Apoptosis was assessed, also in tissue sections, by deoxyuridine triphosphate nick end-labeling (TUNEL) procedure. Results: In heatstroke animals, there were marked EC changes in lungs, spleen, jejunum, kidneys and liver, demonstrated by TEM, as increased cytoplasmic membrane convolutions that included formation of villi projecting into the vessel lumina, and increase in the width of the gaps between ECs. Migration of neutrophils, platelets and erythrocytes through these widened gaps was noted. Weibel-Palade bodies were increased both in size and number in EC of jejunum, lungs and kidneys. This increase correlated with increased endothelial expression of immunologically detectable vWF. TEM also showed that there was increased apoptosis manifested by nuclear chromatin condensation and karyorrhexis and formation of cytoplasmic myelin whorls. Increased EC apoptosis was also observed by TUNEL in the jejunum, lungs, liver and spleen. All these changes were greater in animals with severe HS than in animals with moderate HS, whereas sham heated control animals showed no significant changes. Conclusion: Widespread EC injury with apoptotic cell death is consistent with the hypothesis that the endothelium may play a pathogenic role in heatstroke.

scholarly journals Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Andrea Jurisicova ◽  
Beth M Acton
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Fate ◽  
Embryo Development ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Culture Conditions ◽  
Early Embryo ◽  
Preimplantation Embryo Development

Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under currentin vitroculture conditions. There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions. In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality. An increasing body of evidence indicates that cell fate (i.e. survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo development. The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplantation embryo development. In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate.

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

scholarly journals Novel High-Throughput Deoxyribonuclease 1 Assay

2014 ◽  
Vol 20 (2) ◽  
pp. 202-211 ◽  
Author(s):  
Dae Song Jang ◽  
Narsimha R. Penthala ◽  
Eugene O. Apostolov ◽  
Xiaoying Wang ◽  
Tariq Fahmi ◽  
...  
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Volume ◽  
Dnase I ◽  
Radiation Injuries ◽  
Chemical Library ◽  
Chemical Libraries ◽  
Deoxyribonuclease I ◽  
Highly Sensitive

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z’ ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

Lysophosphatidic acid induces endothelial cell death by modulating the redox environment

2007 ◽  
Vol 292 (3) ◽  
pp. R1174-R1183 ◽  
Author(s):  
Sonia Brault ◽  
Fernand Gobeil ◽  
Audrey Fortier ◽  
Jean-Claude Honoré ◽  
Jean-Sébastien Joyal ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Endothelial Cell ◽  
P38 Mapk ◽  
Lysophosphatidic Acid ◽  
Umbilical Vein ◽  
Oxidant Stress ◽  
Redox Environment ◽  
Endothelial Cell Death

Oxidant stress plays a significant role in hypoxic-ischemic injury to the susceptible microvascular endothelial cells. During oxidant stress, lysophosphatidic acid (LPA) concentrations increase. We explored whether LPA caused cytotoxicity to neuromicrovascular cells and the potential mechanisms thereof. LPA caused a dose-dependent death of porcine cerebral microvascular as well as human umbilical vein endothelial cells; cell death appeared oncotic rather than apoptotic. LPA-induced cell death was mediated via LPA1 receptor, because the specific LPA1 receptor antagonist THG1603 fully abrogated LPA's effects. LPA decreased intracellular GSH levels and induced a p38 MAPK/JNK-dependent inducible nitric oxide synthase (NOS) expression. Pretreatment with the antioxidant GSH precursor N-acetyl-cysteine (NAC), as well as with inhibitors of NOS [ Nω-nitro-l-arginine (l-NNA); 1400W], significantly prevented LPA-induced endothelial cell death (in vitro) to comparable extents; as expected, p38 MAPK (SB203580) and JNK (SP-600125) inhibitors also diminished cell death. LPA did not increase indexes of oxidation (isoprostanes, hydroperoxides, and protein nitration) but did augment protein nitrosylation. Endothelial cytotoxicity by LPA in vitro was reproduced ex vivo in brain and in vivo in retina; THG1603, NAC, l-NNA, and combined SB-203580 and SP600125 prevented the microvascular rarefaction. Data implicate novel properties for LPA as a modulator of the cell redox environment, which partakes in endothelial cell death and ensued neuromicrovascular rarefaction.

scholarly journals Eryptosis and Malaria: New Experimental Guidelines and Re-Evaluation of the Antimalarial Potential of Eryptosis Inducers

2021 ◽  
Vol 11 ◽  
Author(s):  
Coralie Boulet ◽  
Taylah L. Gaynor ◽  
Teresa G. Carvalho
Keyword(s):  
Cell Death ◽  
Molecular Target ◽  
Culture Conditions ◽  
Careful Consideration ◽  
Parasite Development ◽  
Clinical Use ◽  
Accurate Assessment ◽  
Set Up ◽  
Defined Culture

Erythrocytes possess an unusual programmed cell death mechanism termed eryptosis, and several compounds have been previously claimed to induce eryptosis in vitro. Malaria parasites (genus Plasmodium) reside in erythrocytes during the pathogenic part of their life cycle, and the potential of several eryptosis inducers to act as antimalarials has been tested in recent years. However, the eryptosis-inducing capacity of these compounds varies significantly between eryptosis-focused studies and malaria investigations. Here, we investigated the reasons for these discrepancies, we developed a protocol to investigate eryptosis in malaria cultures and we re-evaluated the potential of eryptosis inducers as antimalarials. First, we showed that eryptosis read-out in vitro is dependent on culture conditions. Indeed, conditions that have consistently been used to study eryptosis do not support P. falciparum growth and prime erythrocytes for eryptosis. Next, we defined culture conditions that allow the detection of eryptosis while supporting P. falciparum survival. Finally, we selected six eryptosis-inducers based on their clinical use, molecular target and antimalarial activities, and re-evaluated their eryptosis inducing capacities and their potential as antimalarials. We demonstrate that none of these compounds affect the viability of naïve or P. falciparum-infected erythrocytes in vitro. Nevertheless, three of these compounds impair parasite development, although through a mechanism unrelated to eryptosis and yet to be elucidated. We conclude that careful consideration of experimental set up is key for the accurate assessment of the eryptosis-inducing potential of compounds and their evaluation as potential antimalarials.

Either exogenous or endogenous fibronectin can promote adherence of human endothelial cells

1986 ◽  
Vol 82 (1) ◽  
pp. 263-280
Author(s):  
R.A. Clark ◽  
J.M. Folkvord ◽  
L.D. Nielsen
Keyword(s):  
Endothelial Cells ◽  
Wound Repair ◽  
Cell Types ◽  
Culture Conditions ◽  
Collagen Type Iv ◽  
Human Endothelial Cells ◽  
Small Vessels ◽  
Microvascular Cells

Recently, we have presented evidence that proliferating blood vessels produce and deposit fibronectin in situ during the angiogenesis of wound repair. This report extends these observations by demonstrating that human endothelial cells from both large and small vessels depend on fibronectin for their adherence in vitro. Endothelial cells were grown from human umbilical veins (HUVEC) by the method of Gimbrone and from the microvasculature of human omentum by the method of Kern, Knedler and Eckel. Second-passage cells were plated into microtitre wells that had been coated with 100 micrograms ml-1 of fibronectin, types I and III collagen, type IV collagen or laminin. After a 3-h incubation, adherent cells were solubilized with Zap-Isoton and quantified on a Coulter Counter. Under normal culture conditions HUVEC showed no preference for fibronectin substrates while microvascular cells always demonstrated a striking preference for fibronectin substrates. However, when HUVEC were exposed to 2.5 or 25 micrograms ml-1 of cycloheximide for 4 h before and during the adherence assays, the adherence to fibronectin was 50–200% greater than to types I and III collagen. Immunofluorescence studies showed that while HUVEC expressed a large quantity of surface fibronectin, microvascular cells expressed very little. Metabolic labelling studies confirmed that HUVEC cultures had substantial quantities of fibronectin in their cell layer while microvascular cells did not. In antibody blocking experiments, preincubation of fibronectin-coated surfaces with anti-fibronectin antibodies totally blocked microvascular cell adhesion but only abrogated HUVEC adherence by 50%, presumably since these latter cells were able to deposit additional fibronectin onto the surface during the 3 h assay period. In the presence of cycloheximide anti-fibronectin antibodies totally blocked HUVEC adherence. These results demonstrate that both endothelial cell types rely, at least in part, on fibronectin for adherence in vitro. HUVEC can synthesize, secrete and deposit enough fibronectin for their adherence in vitro, while microvascular cells rely on an exogenous source of fibronectin under these culture conditions. Thus, the increased blood vessel fibronectin observed during angiogenesis in vivo may mediate adherence of the proliferating and migrating endothelial cells.

scholarly journals Differences in the sensitivity of classically and alternatively activated macrophages to TAK1 inhibitor-induced necroptosis

2020 ◽  
Vol 69 (11) ◽  
pp. 2193-2207 ◽  
Author(s):  
Zsófia Varga ◽  
Tamás Molnár ◽  
Anett Mázló ◽  
Ramóna Kovács ◽  
Viktória Jenei ◽  
...  
Keyword(s):  
Cell Death ◽  
Glycogen Synthase ◽  
Therapeutic Option ◽  
Cell Types ◽  
Therapeutic Benefit ◽  
Alternatively Activated Macrophages ◽  
Highly Sensitive ◽  
Anti Inflammatory ◽  
Macrophage Populations

Abstract Controlling the balance of pro-inflammatory M1 versus anti-inflammatory M2 macrophages may have paramount therapeutic benefit in cardiovascular diseases, infections, cancer and chronic inflammation. The targeted depletion of different macrophage populations provides a therapeutic option to regulate macrophage-mediated functions. Macrophages are highly sensitive to necroptosis, a newly described regulated cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like pseudokinase. Antagonists of inhibitors of apoptosis proteins (SMAC mimetics) block RIPK1 ubiquitination, while TGF-activated kinase 1 (TAK1) inhibitors prevent the phosphorylation of RIPK1, resulting in increased necroptosis. We compared the sensitivity of monocyte-derived human M1 and M2 cells to various apoptotic and necroptotic signals. The two cell types were equally sensitive to all investigated stimuli, but TAK1 inhibitor induced more intense necroptosis in M2 cells. Consequently, the treatment of co-cultured M1 and M2 cells with TAK1 inhibitor shifted the balance of the two populations toward M1 dominance. Blockage of either Aurora Kinase A or glycogen synthase kinase 3β, two newly described necroptosis inhibitors, increased the sensitivity of M1 cells to TAK1-inhibitor-induced cell death. Finally, we demonstrated that in vitro differentiated tumor-associated macrophages (TAM-like cells) were as highly sensitive to TAK1 inhibitor-induced necroptosis as M2 cells. Our results indicate that at least two different necroptotic pathways operate in macrophages and the targeted elimination of different macrophage populations by TAK1 inhibitor or SMAC mimetic may provide a therapeutic option to regulate the balance of inflammatory/anti-inflammatory macrophage functions.

Sign in / Sign up

Export Citation Format

Share Document