CUES FOR CELLULAR ASSEMBLY OF VASCULAR ELASTIN NETWORKS

Elastin, a structural protein distributed in the extracellular matrix of vascular tissues is critical to the maintenance of vascular mechanics, besides regulation of cell-signaling pathways involved in injury response and morphogenesis. Thus, congenital absence or disease-mediated degradation of vascular elastin and its malformation within native vessels due to innately poor elastin synthesis by adult vascular cells compromise vascular homeostasis. Current elastin regenerative strategies using tissue engineering principles are limited by the progressive destabilization of tropoelastin mRNA expression in adult vascular cells and the unavailability of scaffolds that can provide cellular cues necessary to up-regulate elastin synthesis and regenerate faithful mimics of native elastin. Since our earlier studies demonstrated the elastogenic utility of hyaluronan (HA)-based cues, we have currently sought to identify a unique set of culture conditions based on HA fragments (0.756-2000 kDa), growth factors (TGF-beta1, IGF-1) and other biomolecules (Cu2+ ions, LOX), which will together enhance synthesis, crosslinking, maturation and fibrous elastin matrix formation by adult SMCs, under both healthy and inflammatory conditions. It was observed that TGF-beta1 (1 ng/mL) together with HA oligomers (0.2 microg/mL) synergistically suppressed SMC proliferation, enhanced tropoelastin (8-fold) and matrix elastin synthesis (5.5-fold), besides improving matrix yield (4.5-fold), possibly by increasing production and activity of lysyl oxidase (LOX). Though addition of IGF-1 alone did not offer any advantage, HA fragments (20-200 kDa) in the presence of IGF-1 stimulated tropoelastin and soluble elastin synthesis more than 2.2-fold, with HMW HA contributing for ~5-fold increase in crosslinked matrix elastin synthesis. Similarly, 0.1 M of Cu2+ ions, alone or together with HA fragments stimulated synthesis of tropoelastin (4-fold) and crosslinked matrix elastin (4.5-fold), via increases in LOX protein synthesis (2.5-fold); these cues also enhanced deposition of mature elastic fibers (~ 1 micron diameter) within these cultures. Interestingly, instead of copper salt addition, even release of Cu2+ ions (~ 0.1 M) from copper nanoparticles (400 ng/mL), concurrent with HA oligomers, promoted crosslinking of elastin into mature matrix, with multiple bundles of highly-crosslinked elastin fiber formation observed (diameter ~ 200-500 nm). These results strongly attest to the potential individual and combined benefits of these cues to faithful elastin matrix regeneration by healthy, patient-derived cells within tissue-engineered vascular constructs. When these cues (TGF-beta1 and HA oligomers) were added to TNF-alpha-stimulated SMC cultures, model cell culture systems mimicking phenotypically-altered cells within aneurysms, they upregulated elastin matrix production, organized elastin protein into fibers, and simultaneously stabilized this matrix by attenuating production of elastolytic enzymes. Similarly these cues also attenuated inflammatory cytokines release within cells isolated from induced-aortic aneurysms in rats, and significantly upregulated elastin synthesis and matrix formation by upregulating LOX and desmosine protein amounts. The cues were also highly effective in organizing the elastin into fibrous matrix structures mimicking the native elastin deposition process. The outcomes of this study might be of tremendous use in optimizing design of HA constructs to modulate vascular healing and matrix synthesis following revascularization, and in enabling repair of elastin networks within diseased or inflammatory (aneurysmal) adult vascular tissues

Elastogenic cues and methods for using same

Disclosed are elastogenic cues that can be utilized to encourage growth and development of elastin-containing cellular constructs. The elastogenic cues include hyaluronan fragments and oligomers, optionally in conjunction with growth factors and/or a source of copper ions. The elastogenic cues can up-regulate elastin matrix synthesis and by vascular smooth muscle cells. In addition to encouraging synthesis of elastin in a cellular matrix and organization into elastic fibers, the elastogenic cues can also stabilize the formed ECM matrix through suppression of elastin-laminin receptor (ELR). In addition, the elastogenic cues can inhibit cell hyper-proliferation (e.g., hyperplasia) common in inflammatory vascular disease

Synergistic Effects of 3D ECM and Chemogradients on Neurite Outgrowth and Guidance: A Simple Modeling and Microfluidic Framework

During nervous system development, numerous cues within the extracellular matrix microenvironment (ECM) guide the growing neurites along specific pathways to reach their intended targets. Neurite motility is controlled by extracellular signal sensing through the growth cone at the neurite tip, including chemoattractive and repulsive cues. However, it is difficult to regenerate and restore neurite tracts, lost or degraded due to an injury or disease, in the adult central nervous system. Thus, it is important to evaluate the dynamic interplay between ECM and the concentration gradients of these cues, which would elicit robust neuritogenesis. Such information is critical in understanding the processes involved in developmental biology, and in developing high-fidelity neurite regenerative strategies post-injury, and in drug discovery and targeted therapeutics for neurodegenerative conditions. Here, we quantitatively investigated this relationship using a combination of mathematical modeling and in vitro experiments, and determined the synergistic role of guidance cues and ECM on neurite outgrowth and turning. Using a biomimetic microfluidic system, we have shown that cortical neurite outgrowth and turning under chemogradients (IGF-1 or BDNF) within 3D scaffolds is highly regulated by the source concentration of the guidance cue and the physical characteristics of the scaffold. A mechanistic-driven partial differential equation model of neurite outgrowth has been proposed, which could also be used prospectively as a predictive tool. The parameters for the chemotaxis term in the model are determined from the experimental data using our microfluidic assay. Resulting model simulations demonstrate how neurite outgrowth was critically influenced by the experimental variables, which was further supported by experimental data on cell-surface-receptor expressions. The model results are in excellent agreement with the experimental findings. This integrated approach represents a framework for further elucidation of biological mechanisms underlying neuronal responses of specialized cell types, during various stages of development, and under healthy or diseased conditions

Mixing Optimization in Grooved Serpentine Microchannels

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Computational fluid dynamics modeling at Reynolds numbers ranging from 10 to 100 was used to characterize the performance of a new type of micromixer employing a serpentine channel with a grooved surface. The new topology exploits the overlap between the typical Dean flows present in curved channels due to the centrifugal forces experienced by the fluids, and the helical flows induced by slanted groove-ridge patterns with respect to the direction of the flow. The resulting flows are complex, with multiple vortices and saddle points, leading to enhanced mixing across the section of the channel. The optimization of the mixers with respect to the inner radius of curvature (Rin) of the serpentine channel identifies the designs in which the mixing index quality is both high (M \u3e 0.95) and independent of the Reynolds number across all the values investigated

ANTIFERTILITY ACTIVITY OF DECASCHISTIA CROTONIFOLIA LEAF EXTRACT ON MALE RATS

Objective: The objective was to investigate the male antifertility effect of Decaschistia crotonifolia leaf extract on male Wistar rats. Methods: The animals were divided into three groups of five animals each. The first group (I) served as control and received normal saline, and remaining Groups II and III were treated with plant extract at a dose of 200 and 400 mg/kg p.o., respectively, for 21 days. Results: Dose-dependent significant decrease in the weight of testes and epididymis was observed. Furthermore, a dose-related reduction in sperm count and motility was observed. A significant decrease in testosterone levels leading to infertility was also observed. Conclusion: The 70% methanolic leaf extract of D. crotonifolia has produced dose-dependent antifertility effect on male rats

Synthesis and Secretome Release by Human Bone Marrow Mesenchymal Stem Cell Spheroids within Three-dimensional Collagen Hydrogels: Integrating Experiments and Modelling

Myocardial infarction results in loss of cardiac cell types, inflammation, extracellular matrix (ECM) degradation, and fibrotic scar. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is being explored as they could differentiate into cardiomyocyte-like cells, integrate into host tissue, and enhance resident cell activity. The ability of these cells to restore lost ECM, remodel the inflammatory scar tissue, and repair the injured myocardium remains unexplored. We here elucidated the synthesis and deposition of ECM (e.g., elastin, sulfated glycosaminoglycans, hyaluronan, collagen type III, laminin, fibrillin, lysyl oxidase, and nitric oxide synthases), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and other secretome (cytokines, chemokines, and growth factors) in adult human BM-MSC spheroid cultures within three-dimensional collagen gels. The roles of species-specific type I collagen and 5-azacytadine were assessed over a 28-day period. Results revealed that human collagen (but not rat-derived) suppressed MSC proliferation and survival, and MSCs synthesized and released a variety of ECM proteins and secretome over the 28 days. Matrix deposition is at least an order of magnitude lower than their release levels at every time point, most possibly due to elevated MMP levels and interleukins with a concomitant decrease in TIMPs. Matrix synthesis over the 28-day period was fitted to a competitive inhibition form of Michaelis-Menten kinetics, and the production and decay rates of ECM, MMPs, and TIMPs, along with the kinetic model parameters quantified. Such an integrated experimental and modelling approach would help elucidate the critical roles of various parameters (e.g., cell encapsulation and delivery vehicles) in stem cell-based transplantation therapies

Biophysical and Biomechanical Properties of Neural Progenitor Cells as Indicators of Developmental Neurotoxicity

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young\u27s modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36 h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p \u3c 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p \u3c 0.01) as well as toxicant concentration (p \u3c 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R-2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes

Sensitivity of Neural Stem Cell Survival, Differentiation and Neurite Outgrowth Within 3D Hydrogels to Environmental Heavy Metals

© 2015 Elsevier Ireland Ltd. We investigated the sensitivity of embryonic murine neural stem cells exposed to 10 pM-10. μM concentrations of three heavy metals (Cd, Hg, Pb), continuously for 14 days within 3D collagen hydrogels. Critical endpoints for neurogenesis such as survival, differentiation and neurite outgrowth were assessed. Results suggest significant compromise in cell viability within the first four days at concentrations ≥10. nM, while lower concentrations induced a more delayed effect. Mercury and lead suppressed neural differentiation at as low as 10 pM concentration within 7 days, while all three metals inhibited neural and glial differentiation by day 14. Neurite outgrowth remained unaffected at lower cadmium or mercury concentrations (≤100. pM), but was completely repressed beyond day 1 at higher concentrations. Higher metal concentrations (≥100. pM) suppressed NSC differentiation to motor or dopaminergic neurons. Cytokines and chemokines released by NSCs, and the sub-cellular mechanisms by which metals induce damage to NSCs have been quantified and correlated to phenotypic data. The observed degree of toxicity in NSC cultures is in the order: lead. \u3e. mercury. \u3e. cadmium. Results point to the use of biomimetic 3D culture models to screen the toxic effects of heavy metals during developmental stages, and investigate their underlying mechanistic pathways

Carboxymethylcellulose hydrogels support cns-derived tumor cell chemotactic migration

The local microenvironment plays an important role in maintaining the dynamics of the extracellular matrix (ECM) and the cell-ECM relationship. ECM is a complex network of molecules with distinct mechanical and biochemical characteristics. When the mechanisms that are in place to maintain ECM homeostasis are deregulated, most likely, this is the onset of cancer. The ECM becomes highly disorganized and the cell-matrix relationship changes, thus promoting alternations in cell mechanisms and metastasis. Medulloblastoma (MB) is one of the most common, malignant pediatric brain tumors in the United States. In order to gain a better understanding between the cell-ECM relationship and cell migratory responses in tumors we investigate 7 different types of ECM proteins via a MB-derived cell line: Poly-D-Lysine (PDL), Matrigel, Laminin, Collagen-1, Fibronectin, a 10% blend of Laminin-Collagen1, a 20% blend of Laminin-Collagen 1 and a new cellulose derived hydrogel, carboxymethylcellulose (CMC). Over time, the average changes in cell morphology, in 2D and 3D, are quantified. Data reveals CMC allows for a cell-ECM relationship typically believed to present in tumors, with cell exhibiting amoeboidal morphology that is believed to indicate the ready-ness of a cell to migrate within a given environment. Further investigation into the CMC hydrogels reveal a polysaccharide that allows for chemotactic study of MB-derived Daoy cells enabling minimal haptotactic migration conducive in the mechanistic study of the cells’ chemotactic behavior. Understanding the cell-ECM relationship provides insight into their interactions and the information obtained can be utilized in studying the natural migratory patterns of cells. CMC allows for such a behavior to be studied along with testing the motility of Daoy cells because the hydrogel provides minimal integrin interaction between the cells and the ECM. This study provides insights into understanding the mechanisms behind tumor-associated migratory patterns via chemokines. The data reflects a new possibility of tackling central nervous system (CNS) diseases by utilizing a platform of natural hydrogels to generate therapies inhibiting metastasis

Bioanalytical Method Development and Validation of Memantine in Human Plasma by High Performance Liquid Chromatography with Tandem Mass Spectrometry: Application to Bioequivalence Study

A simple, sensitive, and rapid HPLC-MS/MS method was developed and validated for quantitative estimation of memantine in human plasma. Chromatography was performed on Zorbax SB-C18 (4.6 × 75 mm, 3.5 μm) column. Memantine (ME) and internal standard Memantine-d6(MED6) were extracted by using liquid-liquid extraction and analyzed by LC-ESI-MS/MS using multiple-reaction monitoring (MRM) mode. The assay exhibited a linear dynamic range of 50.00–50000.00 pg/ml for ME in human plasma. This method demonstrated an intra- and interday precision within the range of 2.1–3.7 and 1.4–7.8%, respectively. Further intra- and interday accuracy was within the range of 95.6–99.8 and 95.7–99.1% correspondingly. The mean recovery of ME and MED6 was 86.07 ± 6.87 and 80.31 ± 5.70%, respectively. The described method was successfully employed in bioequivalence study of ME in Indian male healthy human volunteers under fasting conditions