CEBPB/POU2F2 modulates endothelin 1 expression in prehypertensive SHR vascular smooth muscle cells.

The pathogenesis of hypertension is not fully understood; endothelin 1 (EDN1) is involved in developing essential hypertension. EDN1 can promote vascular smooth muscle cell (VSMC) proliferation or hypertrophy through autocrine and paracrine effects. Proliferating smooth muscle cells in the aorta are 'dedifferentiated' cells that cause increased arterial stiffness and remodeling. Male SHRs had higher aortic stiffness than normal control male WKY rats. Male SHR VSMCs expressed high levels of the EDN1 gene, but endothelial cells did not. Therefore, it is necessary to understand the molecular mechanism of enhanced EDN1 expression in SHR VSMCs. We identified POU2F2 and CEBPB as the main molecules that enhance EDN1 expression in male SHR VSMCs. A promoter activity analysis confirmed that the enhancer region of the Edn1 promoter in male SHR VSMCs was from -1309 to -1279 bp. POU2F2 and CEBPB exhibited an additive role in the enhancer region of the EdnET1 promoter. POU2F2 or CEBPB overexpression sufficiently increased EDN1 expression, and co-transfection with the CEBPB and POU2F2 expression plasmids had additive effects on the activity of the Edn1 promoter and EDN1 secretion level of male WKY VSMCs. In addition, the knockdown of POU2F2 also revealed that POU2F2 is necessary to enhance EDN1 expression in SHR VSMCs. The enhancer region of the Edn1 promoter is highly conserved in rats, mice, and humans. POU2F2 and CEBPB mRNA levels were significantly increased in remodeled human VMSCs. In conclusion, the novel regulation of POU2F2 and CEBPB in VSMCs will help us understand the pathogenesis of hypertension and support the development of future treatments for hypertension.

An Update on Mesoporous Silica Nanoparticle Applications in Nanomedicine.

The efficient and safe delivery of therapeutic drugs, proteins, and nucleic acids are essential for meaningful therapeutic benefits. The field of nanomedicine shows promising implications in the development of therapeutics by delivering diagnostic and therapeutic compounds. Nanomedicine development has led to significant advances in the design and engineering of nanocarrier systems with supra-molecular structures. Smart mesoporous silica nanoparticles (MSNs), with excellent biocompatibility, tunable physicochemical properties, and site-specific functionalization, offer efficient and high loading capacity as well as robust and targeted delivery of a variety of payloads in a controlled fashion. Such unique nanocarriers should have great potential for challenging biomedical applications, such as tissue engineering, bioimaging techniques, stem cell research, and cancer therapies. However, in vivo applications of these nanocarriers should be further validated before clinical translation. To this end, this review begins with a brief introduction of MSNs properties, targeted drug delivery, and controlled release with a particular emphasis on their most recent diagnostic and therapeutic applications.

Mitochondrial transport mediates survival of retinal ganglion cells in affected LHON patients.

The mutations in the genes encoding the subunits of complex I of the mitochondrial electron transport chain are the most common cause of Leber's hereditary optic neuropathy (LHON), a maternal hereditary disease characterized by retinal ganglion cell (RGC) degeneration. The characteristics of incomplete penetrance indicate that nuclear genetic and environmental factors also determine phenotypic expression of LHON. Therefore, further understanding of the role of mutant mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit proteins and nuclear genetic factors/environmental effects in the etiology of LHON is needed. In this study, we generated human-induced pluripotent stem cells (hiPSCs) from healthy control, unaffected LHON mutation carrier, and affected LHON patient. hiPSC-derived RGCs were used to study the differences between affected and unaffected carriers of mitochondrial DNA point mutation m.11778G > A in the MT-ND4 gene. We found that both mutated cell lines were characterized by increase in reactive oxygen species production, however, only affected cell line had increased levels of apoptotic cells. We found a significant increase in retrograde mitochondria and a decrease in stationary mitochondria in the affected RGC axons. In addition, the messenger RNA and protein levels of KIF5A in the LHON-affected RGCs were significantly reduced. Antioxidant N-acetyl-L-cysteine could restore the expression of KIF5A and the normal pattern of mitochondrial movement in the affected RGCs. To conclude, we found essential differences in the mutually dependent processes of oxidative stress, mitochondrial transport and apoptosis between two LHON-specific mutation carrier RGC cell lines, asymptomatic carrier and disease-affected, and identified KIF5A as a central modulator of these differences.

Carboxylated nanodiamond-mediated CRISPR-Cas9 delivery of human retinoschisis mutation into human iPSCs and mouse retina.

Nanodiamonds (NDs) are considered to be relatively safe carbon nanomaterials used for the transmission of DNA, proteins and drugs. The feasibility of utilizing the NDs to deliver CRISPR-Cas9 system for gene editing has not been clearly studied. Therefore, in this study, we aimed to use NDs as the carriers of CRISPR-Cas9 components designed to introduce the mutation in RS1 gene associated with X-linked retinoschisis (XLRS). ND particles with a diameter of 3 nm were functionalized by carboxylation of the surface and covalently conjugated with fluorescent mCherry protein. Two linear DNA constructs were attached to the conjugated mCherry: one encoded Cas9 endonuclease and GFP reporter, another encoded sgRNA and contained insert of HDR template designed to introduce RS1 c.625C>T mutation. Such nanoparticles were successfully delivered and internalized by human iPSCs and mouse retinas, the efficiency of internalization was significantly improved by mixing with BSA. The delivery of ND particles led to introduction of RS1 c.625C>T mutation in both human iPSCs and mouse retinas. Rs1 gene editing in mouse retinas resulted in several pathological features typical for XLRS, such as aberrant photoreceptor structure. To conclude, our ND-based CRISPR-Cas9 delivery system can be utilized as a tool for creating in vitro and in vivo disease models of XLRS. STATEMENT OF SIGNIFICANCE: X-linked retinoschisis (XLRS) is a prevalent hereditary retinal disease, which is caused by mutations in RS1 gene, whose product is important for structural organization of the retina. The recent development of genome editing techniques such as CRISPR-Cas9 significantly improved the prospects for better understanding the pathology and development of treatment for this disease. Firstly, gene editing can allow development of appropriate in vitro and in vivo disease models; secondly, CRISPR-Cas9 can be applied for gene therapy by removing the disease-causative mutation in vivo. The major prerequisite for these approaches is to develop safe and efficient CRISPR-Cas9 delivery system. In this study, we tested specifically modified nanodiamonds for such a delivery system. We were able to introduce Rs1 mutation into the mouse retina and, importantly, observed several XLRS-specific effects.

Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.

P3HT:Bebq2-Based Photovoltaic Device Enhances Differentiation of hiPSC-Derived Retinal Ganglion Cells.

Electric field stimulation is known to affect various cellular processes, including cell fate specification and differentiation, particularly towards neuronal lineages. This makes it a promising therapeutic strategy to stimulate regeneration of neuronal tissues. Retinal ganglion cells (RGCs) is a type of neural cells of the retina responsible for transduction of visual signals from the retina to the brain cortex, and is often degenerated in various blindness-causing retinal diseases. The organic photovoltaic materials such as poly-3-hexylthiophene (P3HT) can generate electric current upon illumination with light of the visible spectrum, and possesses several advantageous properties, including light weight, flexibility and high biocompatibility, which makes them a highly promising tool for electric stimulation of cells in vitro and in vivo. In this study, we tested the ability to generate photocurrent by several formulations of blend (bulk heterojunction) of P3HT (which is electron donor material) with several electron acceptor materials, including Alq3 and bis(10-hydroxybenzo[h]quinolinato)beryllium (Bebq2). We found that the photovoltaic device based on bulk heterojunction of P3HT with Bebq2 could generate photocurrent when illuminated by both green laser and visible spectrum light. We tested the growth and differentiation capacity of human induced pluripotent stem cells (hiPSC)-derived RGCs when grown in interface with such photostimulated device, and found that they were significantly increased. The application of P3HT:Bebq2-formulation of photovoltaic device has a great potential for developments in retinal transplantation, nerve repair and tissue engineering approaches of treatment of retinal degeneration.

Bioactivity and gene expression profiles of hiPSC-generated retinal ganglion cells in MT-ND4 mutated Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is the maternally inherited mitochondrial disease caused by homoplasmic mutations in mitochondrial electron transport chain Complex I subunit genes. The mechanism of its incomplete penetrance is still largely unclear. In this study, we created the patient-specific human induced pluripotent stem cells (hiPSCs) from MT-ND4 mutated LHON-affected patient, asymptomatic mutation carrier and healthy control, and differentiated them into retinal ganglion cells (RGCs). We found the defective neurite outgrowth in affected RGCs, but not in the carrier RGCs which had significant expression of SNCG gene. We observed enhanced mitochondrial biogenesis in affected and carrier derived RGCs. Surprisingly, we observed increased NADH dehydrogenase enzymatic activity of Complex I in hiPSC-derived RGCs of asymptomatic carrier, but not of the affected patient. LHON mutation substantially decreased basal respiration in both affected and unaffected carrier hiPSCs, and had the same effect on spare respiratory capacity, which ensures normal function of mitochondria in conditions of increased energy demand or environmental stress. The expression of antioxidant enzyme catalase was decreased in affected and carrier patient hiPSC-derived RGCs as compared to the healthy control, which might indicate to higher oxidative stress-enriched environment in the LHON-specific RGCs. Microarray profiling demonstrated enhanced expression of cell cycle machinery and downregulation of neuronal specific genes.

Elongation of Axon Extension for Human iPSC-Derived Retinal Ganglion Cells by a Nano-Imprinted Scaffold.

Optic neuropathies, such as glaucoma and Leber's hereditary optic neuropathy (LHON) lead to retinal ganglion cell (RGC) loss and therefore motivate the application of transplantation technique into disease therapy. However, it is a challenge to direct the transplanted optic nerve axons to the correct location of the retina. The use of appropriate scaffold can promote the proper axon growth. Recently, biocompatible materials have been integrated into the medical field, such as tissue engineering and reconstruction of damaged tissues or organs. We, herein, utilized nano-imprinting to create a scaffold mimicking the in vitro tissue microarchitecture, and guiding the axonal growth and orientation of the RGCs. We observed that the robust, long, and organized axons of human induced pluripotent stem cell (iPSC)-derived RGCs projected axially along the scaffold grooves. The RGCs grown on the scaffold expressed the specific neuronal biomarkers indicating their proper functionality. Thus, based on our in vitro culture system, this device can be useful for the neurophysiological analysis and transplantation for ophthalmic neuropathy treatment.

Cell death caused by the synergistic effects of zinc and dopamine is mediated by a stress sensor gene Gadd45b - implication in the pathogenesis of Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) is not completely understood, Zinc (Zn(2+) ) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn(2+) and DA-induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn(2+) treatment and raised to an even higher level by Zn(2+) plus DA treatment. Zn(2+) plus DA treatment-induced PC12 cell death was enhanced when there was over-expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross-talk with Gadd45b during Zn(2+) and DA treatment. The synergistic effects of Zn(2+) and DA on PC12 cell death can be accounted for by an activation of the Gadd45b-induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn(2+) /DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD. Zinc and dopamine are implicated in the degeneration of dopaminergic neurons. We previously demonstrated that zinc and dopamine induced synergistic effects on PC12 cell death. Results from this study show that these synergistic effects can be accounted for by activation of the Gadd45b-induced cell death pathway and inhibition of the p38/JNK survival pathway. We provide in vitro and in vivo evidence to support a novel role for Gadd45b in the pathogenesis of Parkinson's disease.