A stable analogue of throm☐ane A2, 9,11-epithio-11,12-methanothrom☐ane A2, stimulates bone resorption in vitro and osteoclast-like cell formation in mouse marrow culture

Throm☐ane A₂ (TXA₂) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, throm☐ane B₂ (TXB₂). TXA₂ has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA₂ have not yet been proved because of its highly unstable nature.Recently, a chemically stable analogue of TXA₂, 9,11-epithio-11,12-methanothrom☐ane A₂ (STA₂), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E₂ (PGE₂), a well-known bone resorbing agent. STA₂ time-and dose-dependently stimulated the release of ⁴⁵Ca from prelabelled mouse calvariae. Both STA₂ and PGE₂ induced the accumulation of cAMP in mouse calvariae. The TXA₂, agonist. ONO-3708, inhibited STA₂-induced release of ⁴⁵Ca, TXB₂ induced neither bone resolor cAMP accumulation. When mouse marrow cells were cultured with STA₂ for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA₂ added. Again TXB₂ showed no effect on osteoclast-like cell formation. These results indicate a role for TXA₂ in some form of bone resorption.     

Estrogen inhibits osteoclasts formation and bone resorption via microRNA-27a targeting PPARγ and APC

Inhibition of osteoclasts formation and bone resorption by estrogen is very important in the etiology of postmenopausal osteoporosis. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell differentiation and proliferation. Thus, we hypothesized that these regulatory molecules might be implicated in the process of estrogen-decreased osteoclasts formation and bone resorption. Western blot, quantitative real-time polymerase chain reaction, tartrate-resistant acid phosphatase staining, pit formation assay and luciferase assay were used to investigate the role of microRNAs in estrogen-inhibited osteoclast differentiation and bone resorption. We found that estrogen could directly suppress receptor activator of nuclear factor B ligand/macrophage colony-stimulating factor-induced differentiation of bone marrow-derived macrophages into osteoclasts in the absence of stromal cell. MicroRNA-27a was significantly increased during the process of estrogen-decreased osteoclast differentiation. Overexpressing of microRNA-27a remarkably enhanced the inhibitory effect of estrogen on osteoclast differentiation and bone resorption, whereas which were alleviated by microRNA-27a depletion. Mechanistic studies showed that microRNA-27a inhibited peroxisome proliferator-activated receptor gamma (PPARγ) and adenomatous polyposis coli (APC) expression in osteoclasts through a microRNA-27a binding site within the 3′-untranslational region of PPARγ and APC. PPARγ and APC respectively contributed to microRNA-27a-decreased osteoclast differentiation and bone resorption. Taken together, these results showed that microRNA-27a may play a significant role in the process of estrogen-inhibited osteoclast differentiation and function.     

α-Tocotrienol inhibits osteoclastic bone resorption by suppressing RANKL expression and signaling and bone resorbing activity

Vitamin E, an essential nutrient with powerful antioxidant activity, is the mixture of two classes of compounds, tocopherols (TPs) and tocotrienols (TTs). Although TTs exhibit better bone protective activity than α-TP, the underlying mechanism is poorly understood. In this study, we investigated whether α-TT and α-TP can modulate osteoclastic bone resorption. We found that α-TT but not α-TP inhibits osteoclastogenesis in coculture of osteoblasts and bone marrow cells induced by either IL-1 or combined treatment with 1α,25(OH)₂ vitamin D₃ and prostaglandin E₂. In accordance with this, only α-TT inhibited receptor activator of NF-κB ligand (RANKL) expression in osteoblasts. In addition, α-TT but not α-TP inhibited RANKL-induced osteoclast differentiation from precursors by suppression of c-Fos expression, possibly through inhibiting ERK and NF-κB activation. This anti-osteoclastogenic effect was reversed when c-Fos or an active form of NFATc1, a critical downstream of c-Fos during osteoclastogenesis, was overexpressed. Furthermore, only α-TT reduced bone resorbing activity of mature osteoclasts without affecting their survival. Overall, our results demonstrate that α-TT but not α-TP has anti-bone resorptive properties by inhibiting osteoclast differentiation and activation, suggesting that α-TT may have therapeutic value for treating and preventing bone diseases characterized by excessive bone destruction.     

Sanguinarine inhibits osteoclast formation and bone resorption via suppressing RANKL-induced activation of NF-κB and ERK signaling pathways

Sanguinarine is a natural plant extract that has been supplemented in a number of gingival health products to suppress the growth of dental plaque. However, whether sanguinarine has any effect on teeth and alveolar bone health is still unclear. In this study, we demonstrated for the first time that sanguinarine could suppress osteoclastic bone resorption and osteoclast formation in a dose-dependent manner. Sanguinarine diminished the expression of osteoclast marker genes, including TRAP, cathepsin K, calcitonin receptor, DC-STAMP, V-ATPase d2, NFATc1 and c-fos. Further investigation revealed that sanguinarine attenuated RANKL-mediated IκBα phosphorylation and degradation, leading to the impairment of NF-κB signaling pathway during osteoclast differentiation. In addition, sanguinarine also affected the ERK signaling pathway by inhibiting RANKL-induced ERK phosphorylation. Collectively, this study suggested that sanguinarine has protective effects on teeth and alveolar bone health.     

Osthole inhibits osteoclasts formation and bone resorption by regulating NF-κB signaling and NFATc1 activations stimulated by RANKL

Chinese herbal medicine Fructus Cnidii has an outstanding effect on chronic lumbar pain and impotence, also has been used against osteoporosis with high frequency. Yet, the mechanisms of osthole, a derivative of Fructus Cnidii, on osteoclasts remains barely known. In this study, it was found out that osthole (10⁻⁶mol/L, 10⁻⁵mol/L) had the influence of inhibiting osteoclast formation and bone resorptive activities induced by receptor activator of nuclear factor κB ligand (RANKL), rather than affecting the viability of osteoclast-like cells. Furthermore, osthole could also inhibit the messenger RNA expressions of c-Src, tartrate-resistant acid phosphatase, β3-Integrin, matrix metallopeptidase 9, and cathepsin K. The results of the mechanistic study indicated that osthole regulated the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and nuclear factor-κB (NF-κB) activations following the RANKL stimulation. These findings suggested that the inhibitory effects of osthole were associated with restraining the activations of NFATc1 and NF-κB induced by RANKL. Thus osthole can be used as a potential treatment for abnormal bone-resorption related diseases.     

Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL-induced activation of NF-κB and ERK

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.     

Estradiol-17β and nutritional status affect calcium balance, scale and bone resorption, and bone formation in rainbow trout, Oncorhynchus mykiss

The effects of estradiol-17β (E₂) on bone resorption and formation as well as its effects on scale resorption were investigated in rainbow trout in order to elucidate the role of the hormone in calcium mobilization from calcified tissues, and to clarify the importance of scale and bone as calcium reserves during sexual maturation. Furthermore, the effects of nutritional status on calcified tissues and E₂-induced calcium mobilization were studied. In fed as well as fasted rainbow trout, E₂ treatment increased scale osteoclastic activity measured as tartrate-resistant acid phosphatase activity, and reduced scale calcium content, suggesting that E₂ increases scale resorption in both the fed and fasted fish. Using histomorphometry, E₂ treatment was found to decrease pharyngeal bone resorption in fed, but not in fasted rainbow trout. The E₂ effect on rainbow trout bone is consistent with its physiological role in mammals and birds where E₂ has been reported to decrease bone resorption. It appears therefore that rainbow trout protect their skeleton and instead use scales as a source of calcium during E₂-induced calcium mobilization. The formation of pharyngeal bone was decreased by fasting, and the importance of the nutritional status for the activity of the bone cells in rainbow trout is therefore emphasized. Accepted: 8 May 1997     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

β-Carotene inhibits neuroblastoma cell invasion and metastasis in vitro and in vivo by decreasing level of hypoxia-inducible factor-1α

Neuroblastoma is the most prevalent extracranial solid tumor in childhood and has poor clinical outcome due to its high potential for metastasis. Consequently, an understanding of the mechanisms that modulate cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. While β-carotene is a vitamin A precursor that has been shown to exert antioxidant and anticancer effects, the anti-metastatic effects of β-carotene on neuroblastoma cells remain poorly understood. The aim of the present study was to investigate the anti-metastatic effects of β-carotene on highly malignant SK-N-BE(2)C neuroblastoma cells in vitro and in vivo. Treatment of SK-N-BE(2)C cells with β-carotene was found to attenuate the migratory and invasive capabilities of the cells. In addition, the enzymatic activity and expression of matrix metalloproteinase (MMP)-2 was suppressed following β-carotene treatment under both normoxia and hypoxia. To induce metastasis, immunodeficient nude mice were injected with SK-N-BE(2)C cells via the tail vein in vivo. The incidence of liver metastasis and mean tumor volume in mice that were administered β-carotene was decreased compared to controls. Furthermore, mRNA levels of MMPs, membrane-type (MT) 2 MMP and tissue inhibitors of metalloproteinases in liver tumor tissues were also lower following β-carotene treatment. Level of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets, vascular endothelial growth factor and glucose transporter 1 (GLUT1), were lower both in vitro and in vivo following β-carotene treatment. In conclusion, the present study provides the first evidence that β-carotene may represent an effective chemotherapeutic agent by regulating the invasion and metastasis of neuroblastoma via HIF-1α.     

Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.     

The metabolism of γ-aminobutyrate and glucose in potassium ion-stimulated brain tissue in vitro

1. The metabolism of gamma-aminobutyrate (GABA) was investigated in cerebral-cortex slices incubated in glucose-saline medium with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. 2. A rapid release of GABA from the tissue, amounting to 25-30% of the total, was observed on addition of 66m-equiv. of K(+)/1 to the medium; the liberation of other amino acids was relatively small. The effect was apparently specific for K(+); GABA was not released on addition of equivalent amounts of Na(+) or on increasing the respiration rate with 10mm-ammonium chloride. The results show that GABA behaves like the transmitter compounds (acetylcholine, catecholamines) on K(+) stimulation, and therefore now satisfies certain of the criteria required for a transmitter in mammalian brain. 3. The release of GABA from the tissue on addition of K(+) was followed by a slow re-uptake. The rate of uptake of GABA in a medium containing 5.9m-equiv. of K(+)/1 was more than four times that in a medium containing 66m-equiv. of K(+)/1. 4. The concentration of GABA in brain tissue incubated for 1h in a medium containing 66m-equiv. of K(+)/1 was about 50% higher than that observed under normal conditions. 5. There was evidence that exogenous [(14)C]GABA mixed with the endogenous pool(s), since the proportion of the total GABA released on K(+) stimulation was the same, and the specific radioactivity of the liberated GABA was close to that remaining in the tissue, whether the GABA was labelled by [1-(14)C]GABA from the medium or generated in the tissue from [(14)C]glucose. 6. On the basis of these findings and the observations outlined in the preceding papers it was possible to calculate the kinetic constants of GABA metabolism by computer simulation of the results. K(+) stimulation led to a 2.5-fold increase in the flux through the tricarboxylic acid cycle, whereas the flux in the GABA bypath was little affected; as a result the flux through the GABA bypath, which under normal conditions was 8% of that through the tricarboxylic acid cycle, decreased to 3-5%. 7. The metabolism of glutamine was greatly affected by K(+)-stimulation. The ratio of the concentration of glutamine in the slices to that in the medium, which under normal conditions was the smallest among the amino acids investigated, increased from about 17 to 63 in 1h. This effect was attributable partly to an uptake of glutamine from the medium (1.8mumol/h per g) and partly to a net increase in the total amount of glutamine (2.6mumol/h per g). At 1h after the addition of K(+) the net gain of glutamine could be accounted for by the decrease of glutamate. 8. Metabolic compartmentation was evident when brain-cortex slices were incubated in glucose-saline medium and the labelled substrate was [(14)C]GABA, since the specific radioactivity of glutamine exceeded that of glutamate. On addition of K(+) the signs of metabolic compartmentation promptly disappeared: this effect was apparently associated with an increase in the permeability of the compartments containing labelled metabolites derived from [(14)C]GABA. The change in the permeability, however, did not affect all the compartments; when the labelled substrate was [(14)C]glucose the equilibration of labelled amino acids between tissue and medium was similar under normal conditions and in the presence of high concentrations of K(+). 9. The metabolism of [(14)C]glucose was followed by measuring oxygen uptake, respiratory (14)CO(2), and incorporation of (14)C into amino acids. The results showed that K(+) stimulation increased the flux of glucose carbon, both in the glycolytic pathway and in the tricarboxylic acid cycle.     

Assessment of polyembryony in lemon: rescue and in vitro culture of immature embryos

The 27 lemon cultivars analysed could be considered slightly or moderately polyembryonic, with 25 to 43% of seeds being polyembryonic and from 1.3 to 1.6 embryos per seed. On this basis, it is necessary to rescue zygotic embryos at an immature stage. Rescue and in vitro embryo development have been studied in two Citrus limon polyembryonic cultivars. Sucrose (50 and 70 g/l) was combined with Murashige and Skoog and Gamborg’s B5 media and tested for optimal growth response. An important effect of genotype was observed: embryos from cultivar ‘Eureka’ had greater survival, germination percentage, and radical development. While the sucrose concentration in the medium did not have an effect on germination, the medium affected the embryo survival and root development of the seedlings, Gamborg’s B5 medium giving the best results. The ability to form plants in vitro was affected by an increase of embryo developmental stage. The germination and seedling height were greater with embryos of seeds collected 135–150 days after anthesis.     

Betacellulin inhibits osteogenic differentiation and stimulates proliferation through HIF-1α

Cellular signaling via epidermal growth factor (EGF) and EGF-like ligands can determine cell fate and behavior. Osteoblasts, which are responsible for forming and mineralizing osteoid, express EGF receptors and alter rates of proliferation and differentiation in response to EGF receptor activation. Transgenic mice over-expressing the EGF-like ligand betacellulin (BTC) exhibit increased cortical bone deposition; however, because the transgene is ubiquitously expressed in these mice, the identity of cells affected by BTC and responsible for increased cortical bone thickness remains unknown. We have therefore examined the influence of BTC upon mesenchymal stem cell (MSC) and pre-osteoblast differentiation and proliferation. BTC decreases the expression of osteogenic markers in both MSCs and pre-osteoblasts; interestingly, increases in proliferation require hypoxia-inducible factor-alpha (HIF-α), as an HIF antagonist prevents BTC-driven proliferation. Both MSCs and pre-osteoblasts express EGF receptors ErbB1, ErbB2, and ErbB3, with no change in expression under osteogenic differentiation. These are the first data that demonstrate an influence of BTC upon MSCs and the first to implicate HIF-α in BTC-mediated proliferation.     

Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl− secretion in MDCK cells

Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled alpha, beta, and gamma subunits of epithelial Na(+) channel (ENaC) showed that, although most of the short-circuit current (I (sc)) was amiloride sensitive (AS-I (sc)), there was also an amiloride-insensitive component (NS-I (sc)) due to Cl(-) secretion (Morris and Schafer, J Gen Physiol 120:71-85, 2002). In the present studies, we observed a progressive increase in NS-I (sc) and a corresponding decrease in AS-I (sc) during experiments. There was a significant negative correlation between AS-I (sc) and NS-I (sc) both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-I (sc) could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca(2+)-activated Cl(-) channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-I (sc) by 40-60%, with a proportional decrease in NS-I (sc). Addition of 100 muM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-I (sc) that was followed by a sustained increase of 50-60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl(-) secretion and the proportional fall in ENaC-mediated Na(+) absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl(-) secretion and Na(+) absorption.     

The use of charcoal in in vitro culture – A review

Activated charcoal is commonly used in tissue culture media. Its addition to culture medium may promote or inhibit in vitro growth, depending on species and tissues used. The effects of activated charcoal may be attributed to establishing a darkened environment; adsorption of undesirable/inhibitory substances; adsorption of growth regulators and other organic compounds, or the release of growth promoting substances present in or adsorbed by activated charcoal.     

β-Methylamino-L-alanine-induced protein aggregation in vitro and protection by L-serine

The cyanobacterial non-protein amino acid α-amino-β-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system x_c^_ and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA.

     

Hydralazine modifies Aβ fibril formation and prevents modification by lipids in vitro

Lipid oxidative damage and amyloid β (Aβ) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aβ misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aβ production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aβ misfolding and (2) Aβ protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aβ aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aβ, and Aβ was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aβ and also did not prevent Aβ aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aβ formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aβ misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.     

Methotrexate chemotherapy–induced damages in bone marrow sinusoids: An in vivo and in vitro study

Chemotherapeutic agents are very well evident extrinsic stimuli for causing damage to endothelial cells. Methotrexate is an antimetabolite commonly used to treat solid tumours and paediatric cancers. However, studies on the effect(s) of methotrexate on bone marrow microvascular system are inadequate. In the current study, we observed a significant bone marrow microvascular dilation following methotrexate therapy in rats, accompanied by apoptosis induction in bone marrow sinusoidal endothelial cells, and followed by recovery of bone marrow sinusoids associated with increased proliferation of remaining bone marrow sinusoidal endothelial cells. Our in vitro studies revealed that methotrexate is cytotoxic for cultured sinusoidal endothelial cells and can also induce apoptosis which is associated with upregulation of expression ratio of Bax and Bcl-2 genes and Bax/Bcl-2 expression ratio. Furthermore, it was shown that methotrexate can negatively affect proliferation of cultured sinusoidal endothelial cells and also inhibit their abilities of migration and formation of microvessel like tubes. The data from this study indicates that methotrexate can cause significant bone marrow sinusoidal endothelium damage in vivo and induce apoptosis and inhibit proliferation, migration and tube-forming abilities of sinusoidal endothelial cells in vitro.