Intranasal Delivery of Cell-Penetrating Therapeutic Peptide Enhances Brain Delivery, Reduces Inflammation, and Improves Neurologic Function in Moderate Traumatic Brain Injury.

Following traumatic brain injury (TBI), secondary brain damage due to chronic inflammation is the most predominant cause of the delayed onset of mood and memory disorders. Currently no therapeutic approach is available to effectively mitigate secondary brain injury after TBI. One reason is the blood-brain barrier (BBB), which prevents the passage of most therapeutic agents into the brain. Peptides have been among the leading candidates for CNS therapy due to their low immunogenicity and toxicity, bioavailability, and ease of modification. In this study, we demonstrated that non-invasive intranasal (IN) administration of KAFAK, a cell penetrating anti-inflammatory peptide, traversed the BBB in a murine model of diffuse, moderate TBI. Notably, KAFAK treatment reduced the production of proinflammatory cytokines that contribute to secondary injury. Furthermore, behavioral tests showed improved or restored neurological, memory, and locomotor performance after TBI in KAFAK-treated mice. This study demonstrates KAFAK's ability to cross the blood-brain barrier, to lower proinflammatory cytokines in vivo, and to restore function after a moderate TBI.

Copper-Cystine Biohybrid-Embedded Nanofiber Aerogels Show Antibacterial and Angiogenic Properties.

Copper-cystine-based high aspect ratio structures (CuHARS) possess exceptional physical and chemical properties and exhibit remarkable biodegradability in human physiological conditions. Extensive testing has confirmed the biocompatibility and biodegradability of CuHARS under diverse biological conditions, making them a viable source of essential Cu2+. These ions are vital for catalyzing the production of nitric oxide (NO) from the decomposition of S-nitrosothiols (RSNOs) found in human blood. The ability of CuHARS to act as a Cu2+ donor under specific concentrations has been demonstrated in this study, resulting in the generation of elevated levels of NO. Consequently, this dual function makes CuHARS effective as both a bactericidal agent and a promoter of angiogenesis. In vitro experiments have shown that CuHARS actively promotes the migration and formation of complete lumens by redirecting microvascular endothelial cells. To maximize the benefits of CuHARS, they have been incorporated into biomimetic electrospun poly(ε-caprolactone)/gelatin nanofiber aerogels. Through the regulated release of Cu2+ and NO production, these channeled aerogels not only provide antibacterial support but also promote angiogenesis. Taken together, the inclusion of CuHARS in biomimetic scaffolds could hold great promise in revolutionizing tissue regeneration and wound healing.

Evolving therapeutic interventions for the management and treatment of Alzheimer's disease.

Alzheimer's Disease (AD) patients experience diverse symptoms, including memory loss, cognitive impairment, behavioral abnormalities, mood changes, and mental issues. The fundamental objective of this review is to discuss novel therapeutic approaches, with special emphasis on recently approved marketed formulations for the treatment of AD, especially Aducanumab, the first FDA approved moiety that surpasses the blood-brain barrier (BBB) and reduces amyloid plaques in the brain, thereby reducing associated cognitive decline. However, it is still in the phase IV trial and is to be completed by 2030. Other drugs such as lecanemab are also under clinical trial and has recently been approved by the FDA and is also discussed here. In this review, we also focus on active and passive immunotherapy for AD as well as several vaccines, such as amyloid-beta epitope-based vaccines, amyloid-beta DNA vaccines, and stem cell therapy for AD, which are in clinical trials. Furthermore, ongoing pre-clinical trials associated with AD and other novel strategies such as curcumin-loaded nanoparticles, Crispr/ cas9, precision medicine, as well as some emerging therapies like anti-sense therapy are also highlighted. Additionally, we discuss some off-labeled drugs like non-steroidal anti-inflammatory drugs (NSAID), anti-diabetic drugs, and lithium, which can manage symptoms of AD and different non-pharmacological approaches are also covered which can help to manage AD. In summary, we have tried to cover all the therapeutic interventions which are available for the treatment and management of AD under sections approved, clinical phase, pre-clinical phase or futuristic interventions, off-labelled drugs, and non-pharmacological interventions for AD, offering positive findings and well as challenges that remain.

Ion-Selective Membrane-Coated Graphene-Hexagonal Boron Nitride Heterostructures for Field-Effect Ion Sensing.

An intrinsic ion sensitivity exceeding the Nernst-Boltzmann limit and an sp 2 -hybridized carbon structure make graphene a promising channel material for realizing ion-sensitive field-effect transistors with a stable solid-liquid interface under biased conditions in buffered salt solutions. Here, we examine the performance of graphene field-effect transistors coated with ion-selective membranes as a tool to selectively detect changes in concentrations of Ca2+, K+, and Na+ in individual salt solutions as well as in buffered Locke's solution. Both the shift in the Dirac point and transconductance could be measured as a function of ion concentration with repeatability exceeding 99.5% and reproducibility exceeding 98% over 60 days. However, an enhancement of selectivity, by about an order magnitude or more, was observed using transconductance as the indicator when compared to Dirac voltage, which is the only factor reported to date. Fabricating a hexagonal boron nitride multilayer between graphene and oxide further increased the ion sensitivity and selectivity of transconductance. These findings incite investigating ion sensitivity of transconductance in alternative architectures as well as urge the exploration of graphene transistor arrays for biomedical applications.

Negative Feedback Role of Astrocytes in Shaping Excitation in Brain Cell Co-cultures.

Glial cells play an important role in maintaining neuronal homeostasis and may thus influence excitability in epileptogenesis. These cells in the brain have glutamate (Glu) transporters, which remove this neurotransmitter from the extracellular space. Lack of negative (-) feedback makes local neuronal circuits more excitable and potentially contributing to epileptogenic phenomena. In this study, the role of glial cells in providing (-) feedback is shown through different models of brain cells in culture imaged for intracellular calcium concentration [(Ca2+)i]. Moreover, here we study the individual cells by putting them in categories. Neuronal networks with high and low (-) feedback were established by using anti-mitotics to deplete glial cells. Separate stimuli with very low subthreshold concentrations of Glu (250-750 nM) were added to cultures to test if the order of stimulations matter in regard to calcium dynamics outcomes. Additionally, KCl and ATP were used to stimulate glial cells. We found that for cultures high in (-) feedback, order of the stimulus was not important in predicting cellular responses and because of the complexity of networks in low (-) feedback cultures the order of stimulus matters. As an additional method for analysis, comparison of high (-) feedback cultures, and pure astrocytes was also considered. Glial cells in pure astrocyte cultures tend to be larger in size than glial cells in high (-) feedback cultures. The potential effect of (-) feedback at the blood brain barrier (BBB) was also considered for the inflammatory responses of nitric oxide (NO) production and [Ca2+]i regulation using brain microvascular endothelial cells (BMVECs). The inflammatory and calcium signaling pathways both indicate the negative feedback role of astrocytes, poised between the BBB and structures deeper within the brain, where neuronal synapses are homeostatically maintained by glial uptake of neurotransmitters.

Efficient LRP1-Mediated Uptake and Low Cytotoxicity of Peptide L57 In Vitro Shows Its Promise as CNS Drug Delivery Vector.

Although an abundance of drug candidates exists which are aimed at the remediation of central nervous system (CNS) disorders, the utility of some are severely limited by their inability to cross the blood brain barrier. Potential drug delivery systems such as the Angiopep family of peptides have shown modest potential; however, there is a need for novel drug delivery candidates that incorporate peptidomimetics to enhance the efficiency of transcytosis, specificity, and biocompatibility. Here, we report on the first in vitro cellular uptake and cytotoxicity study of a peptidomimetic, cationic peptide, L57. It binds to cluster 4 of the low-density lipoprotein receptor-related protein 1 (LRP1) receptor which is expressed in numerous cell types, such as brain endothelial cells. We used early-passage-number brain microvascular endothelial cells and astrocytes harvested from rat pup brains that highly express LRP1, to study the uptake of L57 versus Angiopep-7 (A7). Uptake of L57 and A7 showed a concentration-dependent increase, with L57 being taken up to a greater degree than A7 at the same concentration. Additionally, peptide uptake in LRP1-deficient PEA 10 cells had greatly reduced uptake. Furthermore, L57 demonstrated excellent cell viability versus A7, showing promise as a potential drug delivery vector for CNS therapeutics.

Morphological Changes in Astrocytes by Self-Oxidation of Dopamine to Polydopamine and Quantification of Dopamine through Multivariate Regression Analysis of Polydopamine Images.

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

Tagged Halloysite Nanotubes as a Carrier for Intercellular Delivery in Brain Microvascular Endothelium.

Neurological disorders that are characterized by unpredictable seizures affect people of all ages. We proposed the use of nanocarriers such as halloysite nanotubes to penetrate the blood-brain barrier and effectively deliver the payload over an extended time period. These 50-nm diameter tubes are a natural biocompatible nanomaterial available in large quantities. We proved a prolonged gradual drug delivery mechanism by the nanotube encapsulating rhodamine isothiocyanate and then ionomycin into brain microvascular endothelial cells (BMVECs). Through delayed diffusion, the nanotubes effectively delivered the drug to the primary BMVECs without killing them, by binding and penetration in time periods of 1 to 24 h.

Cellulose-based biomaterials integrated with copper-cystine hybrid structures as catalysts for nitric oxide generation.

Bionanocomposite materials were developed from the assembly of polymer-coated copper-cystine high-aspect ratio structures (CuHARS) and cellulose fibers. The coating of the metal-organic materials with polyallylamine hydrochloride (PAH) allows their covalent linkage to TEMPO-oxidized cellulose by means of EDC/NHS. The resulting materials can be processed as films or macroporous foams by solvent casting and lyophilization, respectively. The films show good mechanical behavior with Young's moduli around 1.5 GPa as well as resistance in water, while the obtained foams show an open network of interconnected macropores with average diameters around 130 µm, depending on the concentration of the initial suspension, and compression modulus values around 450 kPa, similar to other reported freeze-dried nanocellulose-based aerogels. Based on these characteristics, the cellulose/PAH-CuHARS composites are promising for potential biomedical applications as implants or wound dressing materials. They have proved to be effective in the decomposition of low molecular weight S-nitrosothiols (RSNOs), similar to those existing in blood, releasing nitric oxide (NO). This effect is attributed to the presence of copper in the crystalline structure of the CuHARS building unit, which can be gradually released in the presence of redox species like ascorbic acid, typically found in blood. The resulting biomaterials can offer the interesting properties associated with NO, like antimicrobial activity as preliminary tests showed here with Escherichia coli and Staphylococcus epidermidis. In the presence of physiological concentration of RSNOs the amount of generated NO (around 360 nM) is not enough to show bactericidal effect on the studied bacteria, but it could provide other properties inherent to NO even at low concentration in the nM range like anti-inflammatory and anti-thrombotic effects. The cytotoxic effect recorded of the films on rat brain endothelial cells (BMVECs) is least significant and proves them to be friendly enough for further biological studies.

The Immunomodulatory Potential of Copper and Silver Based Self-Assembled Metal Organic Biohybrids Nanomaterials in Cancer Theranostics.

Copper high aspect ratio structures (CuHARS) and silver cystine nanoparticles (AgCysNPs) are two unique micro/nano particles under study here that show extensive anti-cancer effects on a glioma tumor cell line. These micro/nano particles have shown potent toxicity in the presence of inflammatory stimulus (combination of tumor necrosis factor, [TNF] and lipo-polysaccharide, LPS). CuHARS with a concentration of 20 µg/ml uniquely increased the catalytic generation of nitric oxide (NO), an important contributor in the immune system. This NO was generated in a cell culture tumor microenvironment (TME) in the presence of 25 µM S-nitrosothiol (cysteine-NO) and the inflammatory stimulus. CuHARS increased the NO production by 68.75% when compared to untreated glioma cells with CysNO and inflammatory stimulus. The production of NO was significantly higher under similar circumstances in the case of normal primary structural cells like brain microvascular endothelial cells (BMVECs). The production of NO by BMVECs went up by 181.25% compared to glioma cells. This significant increase in the NO concentration could have added up to tumorigenesis but the anti-cancer effect of CuHARS was prominent enough to lower down the viability of glioma cells by approximately 20% and increased the metabolism of structural cells, BMVECs by approximately 200%. The immunomodulatory effect of NO in the TME under these circumstances in the presence of the novel micro/nano material, CuHARS has risen up compared to the effect of inflammatory stimulus alone. The potency and specific nature of these materials toward tumor cells may make them suitable candidates for cancer treatment. Successive treatment of CuHARS to glioma cells also proved to be an effective approach considering the decrease in the total count of cells by 11.84 fold in case of three successive treatments compared to a single dose which only decreased the cell count by 2.45 fold showing the dose-dependent increasing toxicity toward glioma cells. AgCysNPs are another potent nanomaterial which also proved its significant toxic nature toward tumor cell lines as demonstrated here, but their immunomodulatory response is still unclear and needs to be explored further.

Self-Assembled Metal-Organic Biohybrids (MOBs) Using Copper and Silver for Cell Studies.

The novel synthesis of metal-containing biohybrids using self-assembly methods at physiological temperatures (37 °C) was compared for copper and silver using the amino acid dimer cystine. Once assembled, the copper containing biohybrid is a stable, high-aspect ratio structure, which we call CuHARS. Using the same synthesis conditions, but replacing copper with silver, we have synthesized cystine-capped silver nanoparticles (AgCysNPs), which are shown here to form stable colloid solutions in contrast to the CuHARS, which settle out from a 1 mg/mL solution in 90 min. Both the copper and silver biohybrids, as synthesized, demonstrate very low agglomeration which we have applied for the purpose of applications with cell culture methods, namely, for testing as anti-cancer compounds. AgCysNPs (1000 ng/mL) demonstrated significant toxicity (only 6.8% viability) to glioma and neuroblastoma cells in vitro, with concentrations as low as 20 ng/mL causing some toxicity. In contrast, CuHARS required at least 5 µg/mL. For comparative purposes, silver sulfate at 100 ng/mL decreased viability by 52% and copper sulfate at 100 ng/mL only by 19.5% on glioma cells. Using these methods, the novel materials were tested here as metal-organic biohybrids (MOBs), and it is anticipated that the functionalization and dynamics of MOBs may result in building a foundation of new materials for cellular applications, including cell engineering of both normal and diseased cells and tissue constructs.

Early glioma is associated with abnormal electrical events in cortical cultures.

The prodromal stages of some neurological diseases have a distinct electrical profile which can potentially be leveraged for early diagnosis, predicting disease recurrence, monitoring of disease progression, and better understanding of the disease pathology. Gliomas are tumors that originate from glial cells present in the brain and spinal cord. Healthy glial cells support normal neuronal function and play an important role in modulating the regular electrical activity of neurons. However, gliomas can disrupt the normal electrical dynamics of the brain. Though experimental and clinical studies suggest that glioma and injury to glial cells disrupt electrical dynamics of the brain, whether these disruptions are present during the earliest stages of glioma and glial injury are unclear. The primary aim of this study is to investigate the effect of early in vitro glial pathology (glioma and glial injury in specific) on neuronal electrical activity. In particular, we investigated the effect of glial pathology on neural synchronization: an important phenomenon that underlies several central neurophysiological processes (ScienceDirect, 2018 ). We used two in vitro disease samples: (a) a sample in which cortical cultures were treated with anti-mitotic agents that deplete glial cells and (b) a glioma sample in which healthy cortical cells were cultured with CRL-2303 (an aggressive glioma cell line). Healthy cortical culture samples were used as controls. Cultures were established over a glass dish embedded with microelectrodes that permits simultaneous measurement of extracellular electrical activity from multiple sites of the culture. We observed that healthy cortical cultures produce spontaneous and synchronized oscillations which were attenuated in the absence of glial cells. The presence of glioma was associated with the emergence of two types of "abnormal electrical activity" each with distinct amplitude and frequency profile. Our results indicate that even early stages of glioma and glial injury are associated with distinct changes in neuronal electrical activity. Graphical abstract.

An enzyme-based electrochemical biosensor probe with sensitivity to detect astrocytic versus glioma uptake of glutamate in real time in vitro.

Glutamate, a major excitatory neurotransmitter in the central nervous system, is essential for regulation of thought, movement, memory, and other higher functions controlled by the brain. Dysregulation of glutamate signaling is associated with severe neuropathological conditions, such as epilepsy, and glioma, a form of brain cancer. Glutamate signals are currently detected by several types of neurochemical probes ranging from microdialysis-based to enzyme-based carbon fiber microsensors. However, an important technology gap exists in the ability to measure glutamate dynamics continuously, and in real time, and from multiple locations in the brain, which limits our ability to further understand the involved spatiotemporal mechanisms of underlying neuropathologies. To overcome this limitation, we developed an enzymatic glutamate microbiosensor, in the form of a ceramic-substrate enabled platinum microelectrode array, that continuously, in real time, measures changes in glutamate concentration from multiple recording sites. In addition, the developed microbiosensor is almost four-fold more sensitive to glutamate than enzymatic sensors previously reported in the literature. Further analysis of glutamate dynamics recorded by our microbiosensor in cultured astrocytes (control condition) and glioma cells (pathological condition) clearly distinguished normal versus impaired glutamate uptake, respectively. These results confirm that the developed glutamate microbiosensor array can become a useful tool in monitoring and understanding glutamate signaling and its regulation in normal and pathological conditions. Furthermore, the developed microbiosensor can be used to measure the effects of potential therapeutic drugs to treat a range of neurological diseases.

Integration of a Copper-Containing Biohybrid (CuHARS) with Cellulose for Subsequent Degradation and Biomedical Control.

We previously described the novel synthesis of a copper high-aspect ratio structure (CuHARS) biohybrid material using cystine. While extremely stable in water, CuHARS is completely (but slowly) degradable in cellular media. Here, integration of the CuHARS into cellulose matrices was carried out to provide added control for CuHARS degradation. Synthesized CuHARS was concentrated by centrifugation and then dried. The weighed mass was re-suspended in water. CuHARS was stable in water for months without degradation. In contrast, 25 μg/mL of the CuHARS in complete cell culture media was completely degraded (slowly) in 18 days under physiological conditions. Stable integration of CuHARS into cellulose matrices was achieved through assembly by mixing cellulose micro- and nano-fibers and CuHARS in an aqueous (pulp mixture) phase, followed by drying. Additional materials were integrated to make the hybrids magnetically susceptible. The cellulose-CuHARS composite films could be transferred, weighed, and cut into usable pieces; they maintained their form after rehydration in water for at least 7 days and were compatible with cell culture studies using brain tumor (glioma) cells. These studies demonstrate utility of a CuHARS-cellulose biohybrid for applied applications including: (1) a platform for biomedical tracking and (2) integration into a 2D/3D matrix using natural products (cellulose).

Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique.

Extraction and purification of high quality RNA is a crucial initial step required for a variety of genomic assays. We report a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device. This is performed using a 130 µm diameter stainless steel needle that is amino-linked to dT(15) oligonucleotides for selective hybridization of mRNA. By inserting this probe into the biological sample for only 30 seconds, mRNA is captured with high selectivity and a yield greater than 10 pg per mm of probe length. The probe is then inserted into a lab-on-a-chip device, where the bound poly-adenylated RNA is thermally released and immediately reverse transcribed for subsequent PCR amplification. The insertion of the probe into the microfluidic device is straightforward: the microchannel is formed with an elastomer (PDMS) that, when punctured, will seal around the probe. The specificity and RNA loading capacity of the probes were evaluated using conventional qPCR. This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes. Analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps. Integrating this elegant sample preparation method into a complete lab-on-a-chip system will substantially enhance the speed and automation of mRNA assays for research and clinical diagnostics.

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 °C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Regulatory effects of the JAK3/STAT1 pathway on the release of secreted phospholipase A2-IIA in microvascular endothelial cells of the injured brain.

BACKGROUND: Secreted phospholipase A2-IIA (sPLA2-IIA) is an inducible enzyme released under several inflammatory conditions. It has been shown that sPLA2-IIA is released from rat brain astrocytes after inflammatory stimulus, and lipopolysaccharide (LPS) and nitric oxide (NO) have been implicated in regulation of this release. Here, brain microvascular endothelial cells (BMVECs) were treated with LPS to uncover whether sPLA2-IIA was released, whether nitric oxide regulated this release, and any related signal mechanisms. METHODS: Supernatants were collected from primary cultures of BMVECs. The release of sPLA2-IIA, and the expression of inducible nitric oxide synthase (iNOS), phospho-JAK3, phospho-STAT1, total JAK3 and STAT1, ß-actin, and bovine serum albumin (BSA) were analyzed by Western blot or ELISA. NO production was calculated by the Griess reaction. sPLA2 enzyme activity was measured with a fluorometric assay. Specific inhibitors of NO (L-NAME and aminoguanidine, AG), JAK3 (WHI-P154,WHI), STAT1 (fludarabine, Flu), and STAT1 siRNA were used to determine the involvement of these molecules in the LPS-induced release of sPLA2-IIA from BMVECs. Nuclear STAT1 activation was tested with the EMSA method. The monolayer permeability of BMVECs was measured with a diffusion assay using biotinylated BSA. RESULTS: Treatment of BMVECs with LPS increased the release of sPLA2-IIA and nitrite into the cell culture medium up to 24 h. Pretreatment with an NO donor, sodium nitroprusside, decreased LPS-induced sPLA2-IIA release and sPLA2 enzyme activity, and enhanced the expression of iNOS and nitrite generation after LPS treatment. Pretreatment with L-NAME, AG, WHI-P154, or Flu notably reduced the expression of iNOS and nitrite, but increased sPLA2-IIA protein levels and sPLA2 enzyme activity. In addition, pretreatment of the cells with STAT1 siRNA inhibited the phosphorylation of STAT1, iNOS expression, and nitrite production, and enhanced the release of sPLA2-IIA. Pretreatment with the specific inhibitors of NOS, JAK2, and STAT3 decreased the permeability of BMVECs. In contrast, inhibition of sPLA2-IIA release increased cell permeability. These results suggest that sPLA2-IIA expression is regulated by the NO-JAK3-STAT1 pathway. Importantly, sPLA2-IIA augmentation could protect the LPS-induced permeability of BMVECs. CONCLUSION: Our results demonstrate the important action of sPLA2-IIA in the permeability of microvascular endothelial cells during brain inflammatory events. The sPLA2 and NO pathways can be potential targets for the management of brain MVEC injuries and related inflammation.

Activation of the sonic hedgehog signaling controls human pulmonary arterial smooth muscle cell proliferation in response to hypoxia.

The hedgehog signal pathway plays a crucial role in the angiogenesis and vascular remodeling. However, the function of this pathway in the pulmonary vascular smooth cell proliferation in response to hypoxia remains unknown. In this study, we have demonstrated that the main components of the hedgehog pathway, including sonic hedgehog (SHH), patched1 (PTCH1), smoothened (SMO), GLI and hypoxia-inducible factor 1 (HIF1) are expressed in the human pulmonary arterial smooth muscle cells (HPASMCs). Interestingly, hypoxia significantly enhanced the expression of SHH and HIF1, facilitated the translocation of GLI1 into the nuclei, and promoted the proliferation of HPASMCs. Furthermore, direct activation of the SHH pathway through incubation with the purified recombinant human SHH or with purmorphamine and SAG, two Smo agonists, also enhanced the proliferation of HPASMCs. Importantly, the treatment with anti-SHH and anti-HIF1 antibodies or cyclopamine, a specific SMO inhibitor, markedly inhibited the nuclear translocation of GLI1 and cell proliferation in the HPASMCs induced by hypoxia and activation of the SHH pathway. Moreover, the treatment with cyclopamine increased apoptosis in the hypoxic HPASMCs. These data strongly demonstrate for the first time that the SHH signaling plays a crucial role in the regulation of HPASMC growth in response to hypoxia.

Hypoxic preconditioning suppresses group III secreted phospholipase A2-induced apoptosis via JAK2-STAT3 activation in cortical neurons.

Our previous studies show that group III secreted phospholipases A(2) (sPLA(2)s III) induces extensive neuronal apoptosis in brain cortical cultures. However, the molecular mechanisms underlying sPLA(2) III-induced neuronal injury/death are still unknown. Also it is not clear whether hypoxic pre-conditioning (HPC) is able to protect neurons from the sPLA(2) III insult. In this report, we demonstrate that sPLA(2) III significantly decreased production of Bcl-xl and the ratio of Bcl-xl/Bax, and increased expression of Bax, cleaved caspase 3, and cleaved alpha-Fodrin in primary neuronal culture. HPC prevented the sPLA(2) III-induced decreases in production of Bcl-xl and the ratio of Bcl-xl/Bax, and increases in expression of Bax, cleaved caspase 3, and alpha-Fodrin. However, the HPC-produced neuronal protection was eliminated or attenuated by AG490, rapamycin, and STAT3 shRNA. Our results suggest that sPLA(2) III-induced neuronal apoptosis is likely because of its alterations in expression and activity of Bcl-xl, Bax, caspase 3, and its target gene fodrin; and that HPC-produced neuroprotection against the sPLA(2) III toxicity is mediated via JAK-STAT signal pathways that regulate the expression of Bcl-xl, Bax, and cleaved caspase 3 in cultured cortical neurons.

Porous biocompatible three-dimensional scaffolds of cellulose microfiber/gelatin composites for cell culture.

Physiological tissues, including brain and other organs, have three-dimensional (3-D) aspects that need to be supported to model them in vitro. Here we report the use of cellulose microfibers combined with cross-linked gelatin to make biocompatible porous microscaffolds for the sustained growth of brain cell and human mesenchymal stem cells (hMSCs) in 3-D structure. Live imaging using confocal microscopy indicated that 3-D microscaffolds composed of gelatin or cellulose fiber/gelatin both supported brain cell adhesion and growth for 16days in vitro. Cellulose microfiber/gelatin composites containing up to 75% cellulose fibers can withstand a higher mechanical load than gelatin alone, and composites also provided linear pathways along which brain cells could grow compared to more clumped cell growth in gelatin alone. Therefore, the bulk cellulose microfiber provides a novel skeleton in this new scaffold material. Cellulose fiber/gelatin scaffold supported hMSCs growth and extracellular matrix formation. hMSCs osteogenic and adipogenic assays indicated that hMSCs cultured in cellulose fiber/gelatin composite preserved the multilineage differentiation potential. As natural, biocompatible components, the combination of gelatin and cellulose microfibers, fabricated into 3-D matrices, may therefore provide optimal porosity and tensile strength for long-term maintenance and observation of cells.