Origins and molecular mechanisms underlying renal vascular development.

Kidneys play a crucial role in maintaining homeostasis within the body, and this function is intricately linked to the vascular structures within them. For vascular cells within the kidney to mature and perform their functions effectively, it is imperative that there is a meticulous and well-coordinated spatial alignment between the nephrons and the intricate network of blood vessels within the organ. This spatial arrangement ensures efficient filtration of blood and regulation of the electrolyte balance, blood pressure, and fluid levels. Additionally, the kidneys play a vital role in the regulation of acid-base balance and the production of hormones involved in erythropoiesis and blood pressure regulation. Thus, the close interaction between the vascular system and the kidney's structural organization is essential for maintaining overall physiological balance and health. This article focuses on the vascular development of the kidneys, summarizing the current understanding of the origin and formation of the renal vasculature, and the crucial molecules involved. By elucidating the cellular and molecular mechanisms governing renal vascular development, this article aims to promote advancements in renal regenerative medicine and provide potential avenues for therapeutic interventions to address kidney disease.

Rapid and Stable Formation Method of Human Astrocyte Spheroid in a High Viscous Methylcellulose Medium and Its Functional Advantages.

Astrocytes, a type of glial cell in the brain, are thought to be functionally and morphologically diverse cells that regulate brain homeostasis. Cell immortalization is a promising technique for the propagation of primary human astrocytes. The immortalized cells retain their astrocytic marker mRNA expression at lower levels than the primary cells. Therefore, improvement of the differentiation status is required. The use of a 3D formation technique to mimic structural tissue is a good strategy for reflecting physiological cell-cell interactions. Previously, we developed a spheroid formation method using highly viscous methyl cellulose (MC) medium. In this study, we applied this formation method to the well-established immortalized human astrocyte cell line HASTR/ci35. Stable HASTR/ci35 spheroids were successfully formed in MC medium, and laminin deposition was detected inside of the spheroids. Their functional markers were enhanced compared to conventional spheroids formed in U-bottom plates. The inflammatory response was moderately sensitive, and the ability to support neurite growth was confirmed. The HASTR/ci35 spheroid in the MC medium demonstrated the differentiation phenotype and could serve as a potent in vitro model for matured astrocytes.

Enhancement and maintenance of hepatic metabolic functions by controlling 3D aggregation of cryopreserved human iPS cell-derived hepatocyte-like cells.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells are potent cells to study individual-specific hepatotoxicity for drug screening test. However, the functions of metabolic enzymes are practically low. Here, we reconstituted stable and compact 3D spheroids of commercially available cryopreserved HLCs by our original spheroid formation method with high viscous methylcellulose medium. 3D formation enhanced the hepatic functions and maintained the functions for 14 days. Especially, the expression of cytochrome P450s was 10- to 100-fold enhanced compared to conventional 2D culture, which is applicable to a typical drug-metabolizing test using liquid chromatograph-tandem mass spectrometer. In conclusion, we successfully formed human HLC spheroid from commercially available cryo-preserved cells, which realized remarkable hepatic maturation by prolonged 3D culture, especially in terms of drug-metabolizing enzymes. Our spheroid formation technology has the potential to make HLC spheroids a potent tool in aspects of pharmaceutical research, such as drug screening and pharmacokinetic studies.

Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins.

Angiogenesis is regulated in coordinated fashion by chemical and mechanical cues acting on endothelial cells (ECs). However, the mechanobiological mechanisms of angiogenesis remain unknown. Herein, we demonstrate a crucial role of blood flow-driven intraluminal pressure (IP) in regulating wound angiogenesis. During wound angiogenesis, blood flow-driven IP loading inhibits elongation of injured blood vessels located at sites upstream from blood flow, while downstream injured vessels actively elongate. In downstream injured vessels, F-BAR proteins, TOCA1 and CIP4, localize at leading edge of ECs to promote N-WASP-dependent Arp2/3 complex-mediated actin polymerization and front-rear polarization for vessel elongation. In contrast, IP loading expands upstream injured vessels and stretches ECs, preventing leading edge localization of TOCA1 and CIP4 to inhibit directed EC migration and vessel elongation. These data indicate that the TOCA family of F-BAR proteins are key actin regulatory proteins required for directed EC migration and sense mechanical cell stretching to regulate wound angiogenesis.

Murine neonatal ketogenesis preserves mitochondrial energetics by preventing protein hyperacetylation.

Ketone bodies are generated in the liver and allow for the maintenance of systemic caloric and energy homeostasis during fasting and caloric restriction. It has previously been demonstrated that neonatal ketogenesis is activated independently of starvation. However, the role of ketogenesis during the perinatal period remains unclear. Here, we show that neonatal ketogenesis plays a protective role in mitochondrial function. We generated a mouse model of insufficient ketogenesis by disrupting the rate-limiting hydroxymethylglutaryl-CoA synthase 2 enzyme gene (Hmgcs2). Hmgcs2 knockout (KO) neonates develop microvesicular steatosis within a few days of birth. Electron microscopic analysis and metabolite profiling indicate a restricted energy production capacity and accumulation of acetyl-CoA in Hmgcs2 KO mice. Furthermore, acetylome analysis of Hmgcs2 KO cells revealed enhanced acetylation of mitochondrial proteins. These findings suggest that neonatal ketogenesis protects the energy-producing capacity of mitochondria by preventing the hyperacetylation of mitochondrial proteins.

A new perfusion culture method with a self-organized capillary network.

A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow.

Vascularized cancer on a chip: The effect of perfusion on growth and drug delivery of tumor spheroid.

Tumor vasculature creates a hostile tumor microenvironment (TME) in vivo and nourishes cancers, resulting in cancer progression and drug resistance. To mimic the biochemical and biomechanical environments of tumors in vitro, several models integrated with a vascular network have been reported. However, the tumor responses to biochemical and biomechanical stimuli were evaluated under static conditions and failed to incorporate the effects of blood flow to tumors. In this study, we present a tumor-on-a-chip platform that enables the evaluation of tumor activities with intraluminal flow in an engineered tumor vascular network. The fibroblasts in the tumor spheroid induced angiogenic sprouts, which constructed a perfusable vascular network in a tumor spheroid. The perfusability of the engineered vascular network was preserved during the culture. Moreover, perfusion for over 24 h significantly increased the proliferation activities of tumor cells and decreased cell death in the spheroid. Drug administration under perfusion condition did not show the dose-dependent effects of anticancer drugs on tumor activities in contrast to the results under static conditions. Our results demonstrate the importance of flow in a vascular network for the evaluation of tumor activities in a drug screening platform.

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target of bone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.

Identification of neuronal and angiogenic growth factors in an in vitro blood-brain barrier model system: Relevance in barrier integrity and tight junction formation and complexity.

We previously demonstrated that the co-cultivation of endothelial cells with neural cells resulted in an improved integrity of the in vitro blood-brain barrier (BBB), and that this model could be useful to evaluate the transport properties of potential central nervous system disease drugs through the microvascular brain endothelial. In this study we have used real-time PCR, fluorescent microscopy, protein arrays and enzyme-linked immunosorbent assays to determine which neural- and endothelial cell-derived factors are produced in the co-culture and improve the integrity of the BBB. In addition, a further improvement of the BBB integrity was achieved by adjusting serum concentrations and growth factors or by the addition of brain pericytes. Under specific conditions expression of angiogenic, angiostatic and neurotrophic factors such as endostatin, pigment epithelium derived factor (PEDF/serpins-F1), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial cell growth factor (VEGF) closely mimicked the in vivo situation. Freeze-fracture analysis of these cultures demonstrated the quality and organization of the endothelial tight junction structures and their association to the two different lipidic leaflets of the membrane. Finally, a multi-cell culture model of the BBB with a transendothelial electrical resistance up to 371 (±15) Ω×cm2 was developed, which may be useful for preliminary screening of drug transport across the BBB and to evaluate cellular crosstalk of cells involved in the neurovascular unit.

Effects of silica and titanium oxide particles on a human neural stem cell line: morphology, mitochondrial activity, and gene expression of differentiation markers.

Several in vivo studies suggest that nanoparticles (smaller than 100 nm) have the ability to reach the brain tissue. Moreover, some nanoparticles can penetrate into the brains of murine fetuses through the placenta by intravenous administration to pregnant mice. However, it is not clear whether the penetrated nanoparticles affect neurogenesis or brain function. To evaluate its effects on neural stem cells, we assayed a human neural stem cell (hNSCs) line exposed in vitro to three types of silica particles (30 nm, 70 nm, and <44 µm) and two types of titanium oxide particles (80 nm and < 44 µm). Our results show that hNSCs aggregated and exhibited abnormal morphology when exposed to the particles at concentrations = 0.1 mg/mL for 7 days. Moreover, all the particles affected the gene expression of Nestin (stem cell marker) and neurofilament heavy polypeptide (NF-H, neuron marker) at 0.1 mg/mL. In contrast, only 30-nm silica particles at 1.0 mg/mL significantly reduced mitochondrial activity. Notably, 30-nm silica particles exhibited acute membrane permeability at concentrations =62.5 µg/mL in 24 h. Although these concentrations are higher than the expected concentrations of nanoparticles in the brain from in vivo experiments in a short period, these thresholds may indicate the potential toxicity of accumulated particles for long-term usage or continuous exposure.

Cell-based in vitro blood-brain barrier model can rapidly evaluate nanoparticles' brain permeability in association with particle size and surface modification.

The possibility of nanoparticle (NP) uptake to the human central nervous system is a major concern. Recent reports showed that in animal models, nanoparticles (NPs) passed through the blood-brain barrier (BBB). For the safe use of NPs, it is imperative to evaluate the permeability of NPs through the BBB. Here we used a commercially available in vitro BBB model to evaluate the permeability of NPs for a rapid, easy and reproducible assay. The model is reconstructed by culturing both primary rat brain endothelial cells and pericytes to support the tight junctions of endothelial cells. We used the permeability coefficient (P(app)) to determine the permeability of NPs. The size dependency results, using fluorescent silica NPs (30, 100, and 400 nm), revealed that the Papp for the 30 nm NPs was higher than those of the larger silica. The surface charge dependency results using Qdots® (amino-, carboxyl-, and PEGylated-Qdots), showed that more amino-Qdots passed through the model than the other Qdots. Usage of serum-containing buffer in the model resulted in an overall reduction of permeability. In conclusion, although additional developments are desired to elucidate the NPs transportation, we showed that the BBB model could be useful as a tool to test the permeability of nanoparticles.

Mycobacterium tuberculosis escapes from the phagosomes of infected human osteoclasts reprograms osteoclast development via dysregulation of cytokines and chemokines.

Spinal tuberculosis is a condition characterized by massive resorption of the spinal vertebrae due to the infection with Mycobacterium tuberculosis (Mtb). However, the pathogenesis of spinal tuberculosis has not been established because it was almost completely eradicated by the establishment of antibiotic treatment in the mid-20th century. In this study, we investigated the inflammatory responses of human multinucleated osteoclasts infected with virulent Mtb strain. We found that the intracellular Mtb infection of multinuclear osteoclasts resulted in the rapid growth of Mtb and an osteolytic response, rather than inflammation. In response to Mtb infection, the mononuclear osteoclast precursors produced proinflammatory cytokines including tumor necrosis factor (TNF)-α, an intrinsic characteristic they share with macrophages. In contrast, highly fused multinucleated osteoclasts incapacitated the production of these cytokines. Instead, the intracellular Mtb inside multinuclear osteoclasts escaped from the endosome/phagosome, leading to a different pattern of osteoclast activation, with the production of chemokines such as CCL5, CCL17, CCL20, CCL22, CCL24, and CCL25. Moreover, intracellular infection with an avirulent Mtb strain resulted in diminished production of these chemokines. These findings indicate that intracellular Mtb infection in multinuclear osteoclasts reprograms osteoclast development via the dysregulation of cytokines and chemokines.

Evaluation of anti-inflammatory drug-conjugated silicon quantum dots: their cytotoxicity and biological effect.

Silicon quantum dots (Si-QDs) have great potential for biomedical applications, including their use as biological fluorescent markers and carriers for drug delivery systems. Biologically inert Si-QDs are less toxic than conventional cadmium-based QDs, and can modify the surface of the Si-QD with covalent bond. We synthesized water-soluble alminoprofen-conjugated Si-QDs (Ap-Si). Alminoprofen is a non-steroid anti-inflammatory drug (NSAID) used as an analgesic for rheumatism. Our results showed that the "silicon drug" is less toxic than the control Si-QD and the original drug. These phenomena indicate that the condensed surface integration of ligand/receptor-type drugs might reduce the adverse interaction between the cells and drug molecules. In addition, the medicinal effect of the Si-QDs (i.e., the inhibition of COX-2 enzyme) was maintained compared to that of the original drug. The same drug effect is related to the integration ratio of original drugs, which might control the binding interaction between COX-2 and the silicon drug. We conclude that drug conjugation with biocompatible Si-QDs is a potential method for functional pharmaceutical drug development.

Roles of chemokine receptor CX3CR1 in maintaining murine bone homeostasis through the regulation of both osteoblasts and osteoclasts.

Chemokines have recently been reported to be involved in pathological bone destruction. However, the physiological roles of chemokines in bone metabolism in vivo have not been well documented. We analyzed the bone phenotypes in Cx3cr1-deficient mice. The mice exhibited slight but significant increases in trabecular and cortical thickness, reduced numbers of osteoclasts and increased rates of osteoid formation. Although the morphometric parameters showed marginal differences, the Cx3cr1-deficient bones showed an elevated expression of Osterix/SP7, which encodes an essential transcriptional factor for osteoblasts, whereas the gene Osteocalcin/Bglap, which encodes a late marker, was downregulated. The levels of transcripts for various osteoclastic markers, such as receptor activator of NF-κB (RANK)/TNFRSF11A, receptor activator of NF-κB ligand (RANKL)/TNFSF11, tartrate-resistant acid phosphatase 5b (TRAP5B)/ACP5B, Cathepsin K(CTSK), MMP3 and MMP13, were significantly decreased in the Cx3cr1-deficient bones. Cultured Cx3cr1-deficient osteoblastic cells showed inverse temporal patterns of osteoblastic marker expression and reduced calcium deposition. Furthermore, in vitro studies and immunofluorescence staining against CX3CR1 and CX3CL1 suggested a role for the CX3CR1-CX3CL1 axis in an early stage of osteoblast differentiation, possibly through their trans and cis interactions. Cultured Cx3cr1-deficient pre-osteoclasts showed impaired differentiation, mainly due to a deficiency of the CD115(+)CD11b(lo) osteoclastogenic population of myeloid-lineage precursors. The treatment of bone-marrow-derived osteoclastic cultures with recombinant CX3CL1 at different time points suggested that the CX3CR1-CX3CL1 axis favors the maintenance of osteoclastic precursors, but not differentiated osteoclasts. These observations uncovered novel roles of the CX3CR1-CX3CL1 axis in the differentiation of both osteoblasts and osteoclasts.

Expression of mRNAs of urocortin and corticotropin-releasing factor receptors in malignant glioma cell lines.

BACKGROUND: Urocortin and corticotropin-releasing factors (CRFs) and their receptors are expressed in many organs, including the central nervous system. In this study, the expression of mRNAs of urocortin 1, 2, 3, and CRF and CRF receptors 1 and 2 in malignant glioma, was examined. MATERIALS AND METHODS: The RNAs of human and rat glioma cell lines were isolated. Transcripts in these cells were analyzed using cDNA. In addition, the effects of proliferative and cytotoxic stimulation by serum supplementation, ionizing radiation, and the antineoplastic agent temozolomide were investigated. RESULTS: Human and rat cells transcribed urocortin. CRF receptors were detected in human glioma cells. When human KNS42 cells were exposed to stimulation, transcription was altered according to the specific condition. CONCLUSION: Expression of mRNAs of urocortin and CRF receptors was confirmed in human glioma cell lines. Although the quantities of transcripts varied with the proliferative and cytotoxic stimulation, the overall transcription pattern was not influenced by these stimuli.

Direct oxygen supply with polydimethylsiloxane (PDMS) membranes induces a spontaneous organization of thick heterogeneous liver tissues from rat fetal liver cells in vitro.

Oxygen is a vital nutrient for growth and maturation of in vitro cells (e.g., adult hepatocytes). We previously demonstrated that direct oxygenation through a polydimethylsiloxane (PDMS) membrane increases the oxygen supply to cell cultures and improves hepatocyte functions. In this study, we removed limits on oxygen supply to fetal rat liver cells through the use of direct oxygenation through a PDMS membrane to investigate in vitro growth and maturation. We chose fetal liver cells because they are considered a feasible source of liver progenitor cells for regenerative medicine therapy due to their highly efficient maturation and proliferation. Cells from 17-day-old pregnant rats were cultured under 5% and 21% oxygen atmospheres. Some cells were first cultured under 5% oxygen, and then switched to a 21% oxygen atmosphere. When oxygen supply was enhanced by a PDMS membrane, the rat fetal liver cells organized into a complex tissue composed of an epithelium of hepatocytes above a mesenchyme-like tissue. The thickness of this supportive tissue was directly correlated to oxygen concentration and was thicker under 5% oxygen. When cultures were switched from 5% to 21% oxygen, lumen-containing structures were formed in the thick mesenchymal-like tissue and the albumin secretion rate increased. In addition, cells adapted their glycolytic activity to the oxygen concentrations. This system promoted the formation of a functional and organized thick tissue suitable for use in regenerative medicine.

Toxicity of nanocrystal quantum dots: the relevance of surface modifications.

With the development of nanotechnology, nanometer-sized products smaller than several 100 nm have been applied for all areas of science and technology. The nanometer-sized products, including carbon nanotubes, fullerene derivatives, and nanocrystals made of various materials, are widely employed as novel tools in various fields, not only in material engineering, electronics, plastics, automobile, aviation, and aerospace industries, but also even in cellular biology, molecular biology, and basic and clinical medical fields. In particular, nanocrystal quantum dots (QDs) have been widely used in biological and medical studies because of their far brighter photoemission and photostability. The physical and chemical properties of QDs have been circumstantially investigated, but little is known about the potential harmful effects of QDs on human health. In addition to the physical and chemical properties of the QDs, their toxicity and biological behavior are generally regulated by three other conditions: (1) the QD core material itself, (2) the surface modifications of the QD, and (3) the external environmental condition of the QDs. We herein report on the in vitro and in vivo toxicity and biological behavior of nanocrystals such as QDs. Accumulating evidence suggests that the QD-capping material, rather than the core metalloid complex, is responsible for the majority of their toxicity and biological activity. For example, molecules covered with a toxic agent showed cytotoxicity, whereas QDs conjugated with biomolecules retained the biological effects of the conjugate.

Deficiency of chemokine receptor CCR1 causes osteopenia due to impaired functions of osteoclasts and osteoblasts.

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1(-/-) mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1(-/-) mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1(-/-) mice. Osteoclastogenesis induced from cultured Ccr1(-/-) bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1(-/-) osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1(-/-) mice. The co-culture of wild-type osteoclast precursors with Ccr1(-/-) osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.

Chemical reactions on surface molecules attached to silicon quantum dots.

This Article describes research on chemical reactions on molecules attached to the surface of silicon quantum dots that have been performed to produce quantum dots with reactive surface functionalities such as diols and epoxides. Characterization of the surface reactions includes NMR and FT-IR studies, and the quantum dots were characterized by transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS). Cytotoxicity and cell viability assay conducted on silicon dots capped with polar molecules indicated low toxicity with quantum dots with more reactive functionalities found to be more toxic. The silicon quantum dots photoluminesce and have been used as a blue chromophore for the biological imaging of cells.