Physicochemical Mechanisms of Protection Offered by Agarose Encapsulation during Cryopreservation of Mammalian Cells in the Absence of Membrane-Penetrating Cryoprotectants.

During freeze/thaw, cells are exposed to mechanical, thermal, chemical, and osmotic stresses, which cause loss of viability and function. Cryopreservation agents such as dimethyl sulfoxide (DMSO) are deployed to minimize freeze/thaw damage. However, there is a pressing need to eliminate DMSO from cryopreservation solutions due to its adverse effects. This is of the highest priority especially for cryopreservation of infusible/transplantable cell therapy products. In order to address this issue, we introduce reversible encapsulation in agarose hydrogels in the presence of the membrane-impermeable cryoprotectant, trehalose, as a viable, safe, and effective cryopreservation method. Our findings, which are supported by IR spectroscopy and differential scanning calorimetry analyses, demonstrate that encapsulation in 0.75% agarose hydrogels containing 10-20% trehalose inhibits mechanical damage induced by eutectic phase change, devitrification, and recrystallization, resulting in post-thaw viability comparable to the gold standard 10% DMSO.

Water-Soluble Luminescent Silicon Nanocrystals by Plasma-Induced Acrylic Acid Grafting and PEGylation.

Luminescent silicon nanocrystals are promising nanomaterials for biomedical applications due to their unique optical properties and biocompatibility. Here, we demonstrate a two-step surface modification approach coupling gas-phase and liquid-phase methods to synthesize PEGylated acrylic acid grafted silicon nanocrystals with near-infrared emission in water and biological media. First, acrylic acid grafted silicon nanocrystals are synthesized by an all-gas-phase approach on a millisecond time scale, omitting high temperature and postpurification processes. Subsequently, room-temperature PEGylation is carried out with these acrylic acid grafted silicon nanocrystals, yielding stable colloidal dispersions in both water and high ionic strength Tyrode's buffer with 20-30 nm hydrodynamic diameters. The PEGylated silicon nanocrystals exhibit photoluminescence in the 650-900 nm near-IR window with quantum yields of ∼30% and ∼13% in deionized water and Tyrode's buffer, respectively, after a 7-day oxidation in water. The surface-functionalized Si NCs exhibit relatively small toxicity to MDA-MB-231 cells at concentrations relevant to bioimaging applications.

Collagen fibril abnormalities in human and mice abdominal aortic aneurysm.

Vascular diseases like abdominal aortic aneurysms (AAA) are characterized by a drastic remodeling of the vessel wall, accompanied with changes in the elastin and collagen content. At the macromolecular level, the elastin fibers in AAA have been reported to undergo significant structural alterations. While the undulations (waviness) of the collagen fibers is also reduced in AAA, very little is understood about changes in the collagen fibril at the sub-fiber level in AAA as well as in other vascular pathologies. In this study we investigated structural changes in collagen fibrils in human AAA tissue extracted at the time of vascular surgery and in aorta extracted from angiotensin II (AngII) infused ApoE-/- mouse model of AAA. Collagen fibril structure was examined using transmission electron microscopy and atomic force microscopy. Images were analyzed to ascertain length and depth of D-periodicity, fibril diameter and fibril curvature. Abnormal collagen fibrils with compromised D-periodic banding were observed in the excised human tissue and in remodeled regions of AAA in AngII infused mice. These abnormal fibrils were characterized by statistically significant reduction in depths of D-periods and an increased curvature of collagen fibrils. These features were more pronounced in human AAA as compared to murine samples. Thoracic aorta from Ang II-infused mice, abdominal aorta from saline-infused mice, and abdominal aorta from non-AAA human controls did not contain abnormal collagen fibrils. The structural alterations in abnormal collagen fibrils appear similar to those reported for collagen fibrils subjected to mechanical overload or chronic inflammation in other tissues. Detection of abnormal collagen could be utilized to better understand the functional properties of the underlying extracellular matrix in vascular as well as other pathologies. STATEMENT OF SIGNIFICANCE: Several vascular diseases including abdominal aortic aneurysm (AAA) are characterized by extensive remodeling in the vessel wall. Although structural alterations in elastin fibers are well characterized in vascular diseases, very little is known about the collagen fibril structure in these diseases. We report here a comprehensive ultrastructural evaluation of the collagen fibrils in AAA, using high-resolution microscopy techniques like transmission electron microscopy (TEM) and atomic force microscopy (AFM). We elucidate how abnormal collagen fibrils with compromised D-periodicity and increased fibril curvature are present in the vascular tissue in both clinical AAA as well as in murine models. We discuss how these abnormal collagen fibrils are likely a consequence of mechanical overload accompanying AAA and could impact the functional properties of the underlying tissue.

Dextranol: An inert xeroprotectant.

Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Desiccation protectants (xeroprotectants) like the polysaccharide dextran are critical for preserving dried protein samples by forming a rigid glass that protects entrapped protein molecules. Stably dried proteins are important for maintaining critical information in clinical samples like blood serum as well as maintaining activity of biologic drug compounds. However, we found that dextran reacts with both dried serum proteins and lyophilized purified proteins during storage, producing high-molecular weight Amadori-product conjugates. These conjugates appeared in a matter of days or weeks when stored at elevated temperatures (37° or 45°C), but also appeared on a timescale of months when stored at room temperature. We synthesized a less reactive dextranol by reducing dextran's anomeric carbon from an aldehyde to an alcohol. Serum samples dried in a dextranol-based matrix protected the serum proteins from forming high-molecular weight conjugates. The levels of four cancer-related serum biomarkers (prostate specific antigen, neuropilin-1, osteopontin, and matrix-metalloproteinase 7) decreased, as measured by immunoassay, when serum samples were stored for one to two weeks in dextran-based matrix. Switching to a dextranol-based xeroprotection matrix slightly reduced the damage to osteopontin and completely stopped any detectable damage during storage in the other three biomarkers when stored for a period of two weeks at 45°C. We also found that switching from dextran to dextranol in a lyophilization formulation eliminates this unwanted reaction, even at elevated temperatures. Dextranol offers a small and easy modification to dextran that significantly improves the molecule's function as a xeroprotectant by eliminating the potential for damaging protein-polysaccharide conjugation.

Transcriptomics Analysis Reveals New Insights into the Roles of Notch1 Signaling on Macrophage Polarization.

Naïve macrophages (Mφ) polarize in response to various environmental cues to a spectrum of cells that have distinct biological functions. The extreme ends of the spectrum are classified as M1 and M2 macrophages. Previously, we demonstrated that Notch1 deficiency promotes Tgf-ß2 dependent M2-polarization in a mouse model of abdominal aortic aneurysm. The present studies aimed to characterize the unique set of genes regulated by Notch1 signaling in macrophage polarization. Bone marrow derived macrophages isolated from WT or Notch1+/- mice (n = 12) were differentiated to Mφ, M1 or M2-phenotypes by 24 h exposure to vehicle, LPS/IFN-γ or IL4/IL13 respectively and total RNA was subjected to RNA-Sequencing (n = 3). Bioinformatics analyses demonstrated that Notch1 haploinsufficiency downregulated the expression of 262 genes at baseline level, 307 genes with LPS/IFN-γ and 254 genes with IL4/IL13 treatment. Among these, the most unique genes downregulated by Notch1 haploinsufficiency included fibromodulin (Fmod), caspase-4, Has1, Col1a1, Alpl and Igf. Pathway analysis demonstrated that extracellular matrix, macrophage polarization and osteogenesis were the major pathways affected by Notch1 haploinsufficiency. Gain and loss-of-function studies established a strong correlation between Notch1 haploinsufficiency and Fmod in regulating Tgf-ß signaling. Collectively, our studies suggest that Notch1 haploinsufficiency increases M2 polarization through these newly identified genes.

Smooth muscle cell-specific Notch1 haploinsufficiency restricts the progression of abdominal aortic aneurysm by modulating CTGF expression.

AIMS: Infiltration of macrophages and apoptosis of vascular smooth muscle cells (VSMCs) promote the development of abdominal aortic aneurysm (AAA). Previously, we demonstrated that global Notch1 deficiency prevents the formation of AAA in a mouse model. Herein, we sought to explore the cell-specific roles of Notch1 in AAA development. METHODS AND RESULTS: Cell-specific Notch1 haploinsufficient mice, generated on Apoe-/- background using Cre-lox technology, were infused with angiotensin II (1000 ng/min/kg) for 28 days. Notch1 haploinsufficiency in myeloid cells (n = 9) prevented the formation of AAA attributed to decreased inflammation. Haploinsufficiency of Notch1 in SMCs (n = 14) per se did not prevent AAA formation, but histoarchitectural traits of AAA including elastin degradation and aortic remodeling, were minimal in SMC-Notch1+/-;Apoe-/- mice compared to Apoe-/- mice (n = 33). Increased immunostaining of the contractile SMC-phenotype markers and concomitant decreased expression of synthetic SMC-phenotype markers were observed in the aortae of SMC-Notch1+/-;Apoe-/- mice. Expression of connective tissue growth factor (CTGF), a matrix-associated protein that modulates the synthetic VSMC phenotype, increased in the abdominal aorta of Apoe-/- mice and in the adventitial region of the abdominal aorta in human AAA. Notch1 haploinsufficiency decreased the expression of Ctgf in the aorta and in vitro cell culture system. In vitro studies on SMCs using the Notch1 intracellular domain (NICD) plasmid, dominant negative mastermind-like (dnMAML), or specific siRNA suggest that Notch1, not Notch3, directly modulates the expression of CTGF. CONCLUSIONS: Our data suggest that lack of Notch1 in SMCs limits dilation of the abdominal aorta by maintaining contractile SMC-phenotype and preventing matrix-remodeling.