Protein Arginine Methyltransferase 5 Contributes to Paclitaxel-Induced Neuropathic Pain by Activating Transient Receptor Potential Vanilloid 1 Epigenetic Modification in Dorsal Root Ganglion.

BACKGROUND: Paclitaxel (PTX), which is a first-line chemotherapy drug used to treat various types of cancers, exhibits peripheral neuropathy as a common side effect that is difficult to treat. Protein arginine methyltransferase 5 (PRMT 5) is a key regulator of the chemotherapy response, as chemotherapy drugs induce PRMT5 expression. However, little is known about the PRMT5-mediated epigenetic mechanisms involved in PTX-induced neuropathic allodynia. METHODS: Sprague-Dawley rats were intraperitoneally given PTX to induce neuropathic pain. Biochemical analyses were conducted to measure the protein expression levels in the dorsal root ganglion (DRG) of the animals. The von Frey test and hot plate test were used to evaluate nociceptive behaviors. RESULTS: PTX increased the PRMT5 (mean difference [MD]: 0.68, 95% confidence interval [CI], 0.88-0.48; P < .001 for vehicle)-mediated deposition of histone H3R2 dimethyl symmetric (H3R2me2s) at the transient receptor potential vanilloid 1 ( Trpv1 ) promoter in the DRG. PRMT5-induced H3R2me2s recruited WD repeat domain 5 (WDR5) to increase trimethylation of lysine 4 on histone H3 (H3K4me3) at Trpv1 promoters, thus resulting in TRPV1 transcriptional activation (MD: 0.65, 95% CI, 0.82-0.49; P < .001 for vehicle) in DRG in PTX-induced neuropathic pain. Moreover, PTX increased the activity of NADPH oxidase 4 (NOX4) (MD: 0.66, 95% CI, 0.81-0.51; P < .001 for vehicle), PRMT5-induced H3R2me2s, and WDR5-mediated H3K4me3 in the DRG in PTX-induced neuropathic pain. Pharmacological antagonism and the selective knockdown of PRMT5 in DRG neurons completely blocked PRMT5-mediated H3R2me2s, WDR5-mediated H3K4me3, or TRPV1 expression and neuropathic pain development after PTX injection. Remarkably, NOX4 inhibition not only attenuated allodynia behavior and reversed the above-mentioned signaling but also reversed NOX4 upregulation via PTX. CONCLUSIONS: Thus, the NOX4/PRMT5-associated epigenetic mechanism in DRG has a dominant function in the transcriptional activation of TRPV1 in PTX-induced neuropathic pain.

Paclitaxel exerts antiplatelet and antithrombotic activities: Additional benefit from use of paclitaxel-coated balloons and -eluting stents in coronary revascularization and prevention of in-stent restenosis.

INTRODUCTION: Paclitaxel is a microtubule-stabilizing drug used to treat several types of cancer, including ovarian and breast cancer. Because of its antiproliferative effect on vascular smooth muscle cells, balloons and stents are coated with paclitaxel for use in coronary revascularization and prevention of in-stent restenosis (ISR). However, mechanisms underlying ISR are complicated. Platelet activation is one of the major causes of ISR after percutaneous coronary intervention. Although the antiplatelet activity of paclitaxel was noted in rabbit platelets, the effect of paclitaxel on platelets remains unclear. This study investigated whether paclitaxel exhibits antiplatelet activity in human platelets. METHODS AND RESULTS: Paclitaxel inhibited platelet aggregation induced by collagen but not that induced by thrombin, arachidonic acid, or U46619, suggesting that paclitaxel is more sensitive to the inhibition of collagen-induced platelet activation. Moreover, paclitaxel blocked collagen receptor glycoprotein (GP) VI downstream signaling molecules, including Lyn, Fyn, PLCγ2, PKC, Akt, and MAPKs. However, paclitaxel did not directly bind to GPVI and cause GPVI shedding, as detected by surface plasmon resonance and flow cytometry, respectively, indicating that paclitaxel may interfere with GPVI downstream signaling molecules, such as Lyn and Fyn. Paclitaxel also prevented granule release and GPIIbIIIa activation induced by collagen and low convulxin doses. Moreover, paclitaxel attenuated pulmonary thrombosis and delayed platelet thrombus formation in mesenteric microvessels without significantly affecting hemostasis. CONCLUSION: Paclitaxel exerts antiplatelet and antithrombotic effects. Thus, paclitaxel may provide additional benefits beyond its antiproliferative effect when used in drug-coated balloons and drug-eluting stents for coronary revascularization and prevention of ISR.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

BACKGROUND: The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. METHODS: Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. RESULTS: PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. CONCLUSIONS: pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

Artesunate as a glycoprotein VI antagonist for preventing platelet activation and thrombus formation.

Platelets play a crucial role on hemostasis and are also involved in cardiovascular diseases, such as heart attack and stroke. Artesunate has been reported to possess multiple biological activities, including antitumor and anti-inflammatory activities. However, its effect on platelet activation remains unclear. Thus, we explored the detailed mechanisms underlying its antiplatelet effect. For the in vitro study, the data indicated that artesunate inhibited platelet aggregation induced by collagen, but not thrombin or U46619, indicating that artesunate may selectively inhibit collagen-mediated platelet activation Artesunate also blocked glycoprotein VI (GPVI) downstream signaling, including Syk, PLCγ2, PKC, Akt, and MAPKs. Moreover, artesunate could compete with collagen for binding to collagen receptor and bind to human recombinant GPVI with a high affinity (KD = 44 nM), indicating that it may directly interfere with GPVI. Artesunate also reduced collagen-induced granule release, calcium mobilization, and GPIIbIIIa activation. For the in vivo study, artesunate markedly prevented pulmonary thrombosis and delayed platelet thrombus formation in mesenteric veins and arteries but had minimal effects on hemostasis. In conclusion, we for the first time demonstrated that artesunate acts as a GPVI antagonist and effectively prevents platelet activation and thrombus formation with minimal risk of bleeding, highlighting its therapeutic potential in cardiovascular diseases.

MicroRNA-489-3p attenuates neuropathic allodynia by regulating oncoprotein DEK/TET1-dependent epigenetic modification in the dorsal horn.

Originally characterized as an oncoprotein overexpressed in many forms of cancer that participates in numerous cellular pathways, DEK has since been well described regarding the regulation of epigenetic markers and transcription factors in neurons. However, its role in neuropathic allodynia processes remain elusive and intriguingly complex. Here, we show that DEK, which is induced in spinal dorsal horn neurons after spinal nerve ligation (SNL), is regulated by miR-489-3p. Moreover, SNL-induced decrease in miR-489-3p expression increased the expression of DEK, which recruited TET1 to the promoter fragments of the Bdnf, Grm5, and Stat3 genes, thereby enhancing their transcription in the dorsal horn. Remarkably, these effects were also induced by intrathecally administering naïve animals with miR-489-3p inhibitor, which could be inhibited by knockdown of TET1 siRNA or DEK siRNA. Conversely, delivery of intrathecal miR-489-3p-mimic into SNL rats attenuated allodynia behavior and reversed protein expression coupled to the promoter segments in the dorsal horn. Thus, a spinal miR-489-3p/DEK/TET1 transcriptional axis may contribute to neuropathic allodynia. These results may provide a new target for treating neuropathic allodynia.

Effect of phenylurea hydroxamic acids on histone deacetylase and VEGFR-2.

A series of phenylurea hydroxamic acids incorporating pharmacophores of inhibitors of HDAC inhibitors and VEGFR-2 has been designed. Most of the compounds show antiproliferative activity comparable to that of Vorinostat and Sorafenib, and better EPC inhibitory activity. Enzymatic assays and Western blotting results indicated that compound 14 not only inhibits HDAC but also has slight VEGFR-2 inhibitory activity. A docking study revealed that the polar hydroxamic acid retains the interaction with HDAC through a zinc ion and also interacts with some residues of the active site of VEGFR-2. Despite 14 displaying a weaker VEGFR-2 activity, a possible route to develop potent HDAC/VEGFR-2 inhibitors is suggested.

Deferoxamine accelerates endothelial progenitor cell senescence and compromises angiogenesis.

Senescence reduces the circulating number and angiogenic activity of endothelial progenitor cells (EPCs), and is associated with aging-related vascular diseases. However, it is very time-consuming to obtain aged cells (~1 month of repeated replication) or animals (~2 years) for senescence studies. Here, we established an accelerated senescence model by treating EPCs with deferoxamine (DFO), an FDA-approved iron chelator. Four days of low-dose (3 µM) DFO induced senescent phenotypes in EPCs, including a senescent pattern of protein expression, impaired mitochondrial bioenergetics, altered mitochondrial protein levels and compromised angiogenic activity. DFO-treated early EPCs from young and old donors (< 35 vs. > 70 years old) displayed similar senescent phenotypes, including elevated senescence-associated ß-galactosidase activity and reduced relative telomere lengths, colony-forming units and adenosine triphosphate levels. To validate this accelerated senescence model in vivo, we intraperitoneally injected Sprague-Dawley rats with DFO for 4 weeks. Early EPCs from DFO-treated rats displayed profoundly senescent phenotypes compared to those from control rats. Additionally, in hind-limb ischemic mice, DFO pretreatment compromised EPC angiogenesis by reducing both blood perfusion and capillary density. DFO thus accelerates EPC senescence and appears to hasten model development for cellular senescence studies.

A novel naphthalimide derivative reduces platelet activation and thrombus formation via suppressing GPVI.

Naphthalimide derivatives have multiple biological activities, including antitumour and anti-inflammatory activities. We previously synthesized several naphthalimide derivatives; of them, compound 5 was found to exert the strongest inhibitory effect on human DNA topoisomerase II activity. However, the effects of naphthalimide derivatives on platelet activation have not yet been investigated. Therefore, the mechanism underlying the antiplatelet activity of compound 5 was determined in this study. The data revealed that compound 5 (5-10 µM) inhibited collagen- and convulxin- but not thrombin- or U46619-mediated platelet aggregation, suggesting that compound 5 is more sensitive to the inhibition of glycoprotein VI (GPVI) signalling. Indeed, compound 5 could inhibit the phosphorylation of signalling molecules downstream of GPVI, followed by the inhibition of calcium mobilization, granule release and GPIIb/IIIa activation. Moreover, compound 5 prevented pulmonary embolism and prolonged the occlusion time, but tended to prolong the bleeding time, indicating that it can prevent thrombus formation but may increase bleeding risk. This study is the first to demonstrate that the naphthalimide derivative compound 5 exerts antiplatelet and antithrombotic effects. Future studies should modify compound 5 to synthesize more potent and efficient antiplatelet agents while minimizing bleeding risk, which may offer a therapeutic potential for cardiovascular diseases.

Ultrasonic microbubble VEGF gene delivery improves angiogenesis of senescent endothelial progenitor cells.

The therapeutic effects of ultrasonic microbubble transfection (UMT)-based vascular endothelial growth factor 165 (VEGF165) gene delivery on young and senescent endothelial progenitor cells (EPCs) were investigated. By UMT, plasmid DNA (pDNA) can be delivered into both young EPCs and senescent EPCs. In the UMT groups, higher pDNA-derived protein expression was found in senescent EPCs than in young EPCs. Consistent with this finding, a higher intracellular level of pDNA copy number was detected in senescent EPCs, with a peak at the 2-h time point post UMT. Ultrasonic microbubble delivery with or without VEGF improved the angiogenic properties, including the proliferation and/or migration activities, of senescent EPCs. Supernatants from young and senescent EPCs subjected to UMT-mediated VEGF transfection enhanced the proliferation and migration of human aortic endothelial cells (HAECs), and the supernatant of senescent EPCs enhanced proliferation more strongly than the supernatant from young EPCs. In the UMT groups, the stronger enhancing effect of the supernatant from senescent cells on HAEC proliferation was consistent with the higher intracellular VEGF pDNA copy number and level of protein production per cell in the supernatant from senescent cells in comparison to the supernatant from young EPCs. Given that limitations for cell therapies are the inadequate number of transplanted cells and/or insufficient cell angiogenesis, these findings provide a foundation for enhancing the therapeutic angiogenic effect of cell therapy with senescent EPCs in ischaemic cardiovascular diseases.

S-Phase Kinase-associated Protein-2 Rejuvenates Senescent Endothelial Progenitor Cells and Induces Angiogenesis in Vivo.

Cell cycle slowdown or arrest is a prominent feature of cellular senescence. S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, is a key regulator of G1/S transition. We investigated whether Skp2 plays a role in the regulation of endothelial progenitor cell (EPC) senescence, which is closely associated with aging-related vasculopathy. Replication-induced senescent EPCs demonstrated more pronounced senescence markers and lower Skp2 levels in comparison with those of their younger counterparts. Depletion of Skp2 induced increases in senescence-associated ß-galactosidase (SA-ßGal) activity and a reduction of telomere length and generated a senescent bioenergetics profile, whereas adenoviral-mediated Skp2 expression reversed the relevant senescence. EPCs isolated from older rats displayed a reduced proliferation rate and increased SA-ßGal activity, both of which were significantly reversed by Skp2 ectopic expression. In addition to reversing senescence, Skp2 also rescued the angiogenic activity of senescent EPCs in the ischemic hind limbs of nude mice. The results revealed that ectopic expression of Skp2 has the potential to rejuvenate senescent EPCs and rescue their angiogenic activity and thus may be pivotal in the development of novel strategies to manage aging-related vascular disease.

Inhibiting MLL1-WDR5 interaction ameliorates neuropathic allodynia by attenuating histone H3 lysine 4 trimethylation-dependent spinal mGluR5 transcription.

ABSTRACT: Mixed lineage leukemia 1 (MLL1)-mediated histone H3 lysine 4 trimethylation (H3K4me3) of a subset of genes has been linked to the transcriptional activation critical for synaptic plasticity, but its potential contribution to neuropathic allodynia development remains poorly explored. Here, we show that MLL1, which is induced in dorsal horn neuron after spinal nerve ligation (SNL), is responsible for mechanical allodynia and increased H3K4me3 at metabotropic glutamate receptor subtype 5 (mGluR5) promoter. Moreover, SNL induced WD (Trp-Asp) repeat domain 5 subunit (WDR5) expression as well as the MLL1-WDR5 interaction accompany with H3K4me3 enrichment and transcription of mGluR5 gene in the dorsal horn in neuropathic allodynia progression. Conversely, WDR5-0103, a novel inhibitor of the MLL1-WDR5 interaction, reversed SNL-induced allodynia and inhibited SNL-enhanced mGluR5 transcription/expression as well as MLL1, WDR5, and H3K4me3 at the mGluR5 promoter in the dorsal horn. Furthermore, disrupting the expression of MLL1 or WDR5 using small interfering RNA attenuated mechanical allodynia and reversed protein transcription/expression and complex localizing at mGluR5 promoter in the dorsal horn induced by SNL. This finding revealed that MLL1-WDR5 complex integrity regulates MLL1 and WDR5 recruitment to H3K4me3 enrichment at mGluR5 promoter in the dorsal horn underlying neuropathic allodynia. Collectively, our findings indicated that SNL enhances the MLL1-WDR5 complex, which facilitates MLL1 and WDR5 recruitment to H3K4me3 enrichment at mGluR5 promoter in spinal plasticity contributing to neuropathic allodynia pathogenesis.

Spinal RNF20-Mediated Histone H2B Monoubiquitylation Regulates mGluR5 Transcription for Neuropathic Allodynia.

To date, histone H2B monoubiquitination (H2Bub), a mark associated with transcriptional elongation and ongoing transcription, has not been linked to the development or maintenance of neuropathic pain states. Here, using male Sprague Dawley rats, we demonstrated spinal nerve ligation (SNL) induced behavioral allodynia and provoked ring finger protein 20 (RNF20)-dependent H2Bub in dorsal horn. Moreover, SNL provoked RNF20-mediated H2Bub phosphorylated RNA polymerase II (RNAPII) in the promoter fragments of mGluR5, thereby enhancing mGluR5 transcription/expression in the dorsal horn. Conversely, focal knockdown of spinal RNF20 expression reversed not only SNL-induced allodynia but also RNF20/H2Bub/RNAPII phosphorylation-associated spinal mGluR5 transcription/expression. Notably, TNF-α injection into naive rats and specific neutralizing antibody injection into SNL-induced allodynia rats revealed that TNF-α-associated allodynia involves the RNF20/H2Bub/RNAPII transcriptional axis to upregulate mGluR5 expression in the dorsal horn. Collectively, our findings indicated TNF-α induces RNF20-drived H2B monoubiquitination, which facilitates phosphorylated RNAPII-dependent mGluR5 transcription in the dorsal horn for the development of neuropathic allodynia.SIGNIFICANCE STATEMENT Histone H2B monoubiquitination (H2Bub), an epigenetic post-translational modification, positively correlated with gene expression. Here, TNF-α participated in neuropathic pain development by enhancing RNF20-mediated H2Bub, which facilitates phosphorylated RNAPII-dependent mGluR5 transcription in dorsal horn. Our finding potentially identified neuropathic allodynia pathophysiological processes underpinning abnormal nociception processing and opens a new avenue for the development of novel analgesics.

Growth Arrest and DNA-damage-inducible Protein 45ß-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats.

BACKGROUND: Growth arrest and DNA-damage-inducible protein 45ß reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage-inducible protein 45ß-dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear. METHODS: Adult male Sprague-Dawley rats (654 out of 659) received a spinal nerve ligation or a sham operation with or without intrathecal application of one of the following: growth arrest and DNA-damage-inducible protein 45ß messenger RNA-targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage-inducible protein 45ß, Ro 25-6981 (an NR2B-bearing N-methyl-D-aspartate receptor antagonist), or KN-93 (a calmodulin-dependent protein kinase II antagonist) were used for behavioral measurements, Western blotting, immunofluorescence, dot blots, detection of unmodified cytosine enrichment at cytosine-phosphate-guanine site, chromatin immunoprecipitation quantitative polymerase chain reaction analysis, and slice recordings. RESULTS: Nerve ligation-enhanced growth arrest and DNA-damage-inducible protein 45ß expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage-inducible protein 45ß expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage-inducible protein 45ß to the voltage-dependent T-type calcium channel 3.2 subunit promoter (n = 6) that decreased expression of and current mediated by the voltage-dependent T-type calcium channel 3.2 subunit (both n = 6). In addition, NR2B-bearing N-methyl-D-aspartate receptors and calmodulin-dependent protein kinase II act in an upstream cascade to increase growth arrest and DNA-damage-inducible protein 45ß expression, hence enhancing demethylation at the voltage-dependent T-type calcium channel 3.2 subunit promoter and up-regulating voltage-dependent T-type calcium channel 3.2 subunit expression. Intrathecal administration of Ro 25-6981, KN-93, or a growth arrest and DNA-damage-inducible protein 45ß-targeting small interfering RNA (n = 6) reversed the ligation-induced enrichment of unmodified cytosine at the voltage-dependent T-type calcium channel 3.2 subunit promoter by increasing the associated 5-formylcytosine and 5-carboxylcytosine levels. CONCLUSIONS: By converting 5-formylcytosine or 5-carboxylcytosine to unmodified cytosine, the NR2B-bearing N-methyl-D-aspartate receptor, calmodulin-dependent protein kinase II, or growth arrest and DNA-damage-inducible protein 45ß pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.

Butein Inhibits Angiogenesis of Human Endothelial Progenitor Cells via the Translation Dependent Signaling Pathway.

Compelling evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs) can contribute to postnatal neovascularization and tumor angiogenesis. EPCs have been shown to play a "catalytic" role in metastatic progression by mediating the angiogenic switch. Understanding the pharmacological functions and molecular targets of natural products is critical for drug development. Butein, a natural chalcone derivative, has been reported to exert potent anticancer activity. However, the antiangiogenic activity of butein has not been addressed. In this study, we found that butein inhibited serum- and vascular endothelial growth factor- (VEGF-) induced cell proliferation, migration, and tube formation of human EPCs in a concentration dependent manner without cytotoxic effect. Furthermore, butein markedly abrogated VEGF-induced vessels sprouting from aortic rings and suppressed microvessel formation in the Matrigel implant assay in vivo. In addition, butein concentration-dependently repressed the phosphorylation of Akt, mTOR, and the major downstream effectors, p70S6K, 4E-BP1, and eIF4E in EPCs. Taken together, our results demonstrate for the first time that butein exhibits the antiangiogenic effect both in vitro and in vivo by targeting the translational machinery. Butein is a promising angiogenesis inhibitor with the potential for treatment of cancer and other angiogenesis-related diseases.

Reduction of connexin43 in human endothelial progenitor cells impairs the angiogenic potential.

Our previous work showed that arsenic trioxide down-regulated Cx43 and attenuated the angiogenic potential of human late endothelial progenitor cells (EPC). However, the relation between Cx43 and angiogenic activity of the EPC remained unclear. In the study, human late EPC were treated with siRNA specific to Cx43 (Cx43siRNA). The expression profiles as well as activity of the treated cells were examined. In parallel, the angiogenic potential of human EPC treated with Cx43siRNA was evaluated using murine hind limb ischemic model. The results showed that, in the EPC treated with Cx43siRNA, the activity of migration, proliferation, and angiogenic potential were attenuated, accompanied by reduction in vascular endothelial growth factor (VEGF) expression. In hind limb ischemia mice, EPC treated with Cx43siRNA lost the therapeutic angiogenic potential. VEGF supplementation partially recovered the activity impaired by Cx43 down-regulation. In conclusion, reduced Cx43 expression per se in the EPC causes decreased expression of VEGF and impaired angiogenic potential of the cells. Prevention of Cx43 reduction is a potential target to maintain the angiogenic potential of the EPC.

PRSS23 is essential for the Snail-dependent endothelial-to-mesenchymal transition during valvulogenesis in zebrafish.

AIMS: Cardiac valve disease is a common cause of congenital heart failure. Cardiac valve development requires a complex regulation of assorted protease activities. Nevertheless, the regulation of these proteases during atrioventricular (AV) valve formation is poorly understood. Previously, PRSS23, a novel vascular protease, is shown to be highly expressed at the AV canal during murine heart development; however, its function remains unknown. In this study, we sought to characterize the functional role of PRSS23 during cardiac valve formation. METHODS AND RESULTS: We used a transgenic zebrafish line with fluorescently labelled vasculature as a tool to study the function of PRSS23. We first cloned the zebrafish prss23 and confirmed its sequence conservation with other vertebrate orthologues. Expression of prss23 was detected in the ventricle, atrium, and AV canal during zebrafish embryonic development. We found that morpholino knockdown of Prss23 inhibited the endothelial-to-mesenchymal transition (EndoMT) at the AV canal. Moreover, in human aortic endothelial cell-based assays, PRSS23 knockdown by short-hairpin RNA not only repressed the transforming growth factor-ß-induced EndoMT, but also reduced Snail transcription, suggesting that Snail signalling is downstream of PRSS23 during EndoMT. We further demonstrated that human PRSS23 and SNAIL could rescue the prss23 morpholino-induced AV canal defect in zebrafish embryos, indicating that the function of PRSS23 in valvulogenesis is evolutionarily conserved. CONCLUSION: We demonstrated for the first time that the initiation of EndoMT in valvulogenesis depends on PRSS23-Snail signalling and that the functional role of PRSS23 during AV valve formation is evolutionarily conserved.

Rapid transformation of protein-caged nanomaterials into microbubbles as bimodal imaging agents.

We present a general method for converting colloidal nanomaterials into microbubbles as ultrasound contrast agents. Protein-caged nanomaterials, made either by self-assembled nanoparticles' protein corona or by fluorescent gold nanoclusters, can be rapidly transformed into microbubbles via a sonochemical route, which promote disulfide cross-linking of cysteine residues between protein-caged nanomaterials and free albumin during acoustic cavitation. The proposed methods yielded microbubbles with multiple functions by adjusting the original nanoparticle/protein mixture. We also showed a new dual-modal imaging agent of fluorescent gold microbubbles in vitro and in vivo, which can hold many potential applications in medical diagnostics and therapy.

Synthesis, characterization, and bioconjugation of fluorescent gold nanoclusters toward biological labeling applications.

Synthesis of ultrasmall water-soluble fluorescent gold nanoclusters is reported. The clusters have a decent quantum yield, high colloidal stability, and can be readily conjugated with biological molecules. Specific staining of cells and nonspecific uptake by living cells is demonstrated.

Activation of endothelial cells to pathological status by down-regulation of connexin43.

AIMS: We investigated the effects of connexin43 (Cx43) down-regulation on endothelial function. METHODS AND RESULTS: We used two different sequences of Cx43-specific small interference RNA (siRNA) to reduce de novo synthesis of Cx43 in human aortic endothelial cells and then examined the expression profiles, proliferation activity and viability, and angiogenic potential. The involvement of mitogen-activated protein kinase signalling pathways was analysed. In parallel, the effect of inhibition of gap-junctional communication by connexin-mimetic peptides was evaluated. During the down-regulation of Cx43 by the siRNA, the cells exhibited impaired gap-junctional communication, proliferation, viability, and angiogenic potential. In addition, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor were up-regulated. Furthermore, c-jun N-terminal kinase (JNK) and its downstream target c-jun were activated, while caspase-3, p38, and extracellular signal-regulated kinase remained unchanged. Inhibition of JNK by SP600125 blocked the siRNA-induced increased expression of PAI-1 and partially recovered the impaired angiogenic potential. Short-term inhibition of Cx43 channels by connexin-mimetic peptides did not activate JNK. CONCLUSION: Down-regulation of Cx43 inhibits gap-junctional communication and activates endothelial cells to pathological status, as characterized by up-regulation of coagulatory molecules and impairment of proliferation, viability, and angiogenesis. The processes are associated with activation of JNK signalling pathways and rectified by inhibition of the activation. These results suggest that inadequate expression of Cx43 per se impairs endothelial function by the activation of stress-activated protein kinase.

beta(2)-glycoprotein I protects J774A.1 macrophages and human coronary artery smooth muscle cells against apoptosis.

beta(2)-Glycoprotein I (beta(2)-GPI) is a plasma glycoprotein with multifactorial relevance to clinical consequences. It was previously indicated that beta(2)-GPI can selectively bind to apoptotic cells. This study was designed to determine the role of beta(2)-GPI in apoptosis. Using an immunohistochemical study, we observed that beta(2)-GPI was co-localized with the apoptotic macrophages and smooth muscle cells (SMCs) of human coronary arteries. The contribution of beta(2)-GPI to apoptotic death was then investigated in vascular cells. Two nitric oxide (NO) donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl penicillamine (SNAP) were used in this study to trigger apoptosis in J774A.1 macrophages and human coronary artery smooth muscle cells (HCASMC). Cell viability was significantly improved in beta(2)-GPI-treated cells. It was also possible to detect a remarkable inhibitory effect by beta(2)-GPI on the NO-induced apoptosis by preventing nuclear shrinkage. Furthermore, the NO-induced apoptosis was associated with increase in caspase-3 activity and in the protein levels of caspase-3, c-Fos, and c-Jun. However, all these apoptosis-related events were inhibited in vascular cells treated with 200 microg/ml beta(2)-GPI. This is the first study to show that beta(2)-GPI may be important in the prevention of apoptosis in vascular cells.