bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellulose hydrogels as delivery vehicle of immunomodulatory mesenchymal stem cells
Authors: Flores Andrea Sofia , Gonzalez Keishla , Domenech Maribella
Published: June 2021

Mesenchymal stem cells (MSCs) are cells that have the capacity of differentiation and specialization in skeletal tissues, and more recently immunomodulatory properties. Upon stimulation MSCs secrete immunosuppressive factors that can benefit patients with inflammatory conditions. To prolong therapeutic benefit of MSCs following implantation, delivery strategies that provide protection from external damage are needed. In this study, we evaluated a cellulose gel as a vehicle for encapsulation and delivery of MSCs. Cellulose is biocompatible and non‐degradable in humans, and the use of hydrogels can control the release of anti‐inflammatory factors. To determine the effectiveness of using cellulose hydrogels as a delivery vehicle and establish the relationship between MSCs and cellulose, MSCs were cultivated into three‐dimensional spheroids in cellulose matrixes of 0.3% and 0.5%. Proliferation was measured by spheroid growth over the course of 10 days and was then tested for the secretion profile of its main immunomodulatory factor, indoleamine‐pyrrole 2,3‐dioxygenase (IDO), after stimulation with IFN‐γ +/−. These results were compared to two‐dimensional controls, where higher secretion of IDO was expected in the three‐dimensional models. Multiple spheroids were formed in each concentration instead of one large spheroid. Proliferation was directly proportional to cellulose concentration, although the difference in spheroid growth between both concentrations was considered insignificant. These results show that cellulose based materials can be used as a matrix for in‐vitro studies and as a delivery vehicle for immunomodulatory MSCs.

Support or Funding Information

Financial support from the National Institute of Health RISE program under grant number 1R25GM127191‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased Endoplasmic Reticulum Amino Peptidase‐1 in Sickle Cell Disease: Role of Hypoxia
Authors: Echevarria‐Lorenzo Nanivette , Sanchez‐Real David , Irizarry Jose , Inostroza‐Nieves Yaritza , Rivera Alicia , Romero Jose
Published: June 2021

Sickle cell disease (SCD) is a genetic disorder caused by a single amino acid substitution in the β‐globin gene that results in hemoglobin polymerization, deformed erythrocytes and endothelial cell activation leading to chronic inflammation, anemia, hypoxemia and vaso‐occlusive crisis (VOC). These events contribute to the development of acute and often severe and debilitating, painful episodes in patients with SCD through unclear mechanisms. In SCD, chronic hypoxia and subacute inflammation contribute to endothelial cell activation that is associated with increased inflammatory cytokine markers such as endothelin‐1 (ET‐1), tumor necrosis factor‐α (TNFα), and TNFα receptor‐1 (TNFR1). Endoplasmic Reticulum Aminopeptidase‐1 (ERAP1) is a ubiquitously expressed zinc‐metallopeptidase involved in regulating immune function and inflammation that cleaves cytokine cell surface receptors such as TNFR1 and plays an important role in the pathophysiology of ankylosing spondylitis. However, the relationship between ERAP1 and SCD is, to the best of our knowledge, unexplored. We first studied the effects of deoxygenation (5% O2) on ERAP1 mRNA expression levels by qRT‐PCR in the human endothelial cell line, EA.hy926. We observed a 3‐fold increase in the expression of ERAP1 after 4 hrs of hypoxia (p<0.05, n=4) and 4‐fold increase after 8 hrs of hypoxia (p<0.05, n=4); events that were associated with a 3.5‐fold increase in the hypoxia‐inducible factor 1‐α mRNA levels at these time points. Consistent with these results, activation of EA.hy926 cells with agonist such as angiotensin II was likewise associated with increased ERAP1 mRNA (p<0.05, n=4). We then studied two sickle transgenic knockout mouse models expressing human sickle hemoglobin, the βSAntilles and Berkeley (BERK) mice. We observed significant increases in ERAP1 mRNA levels (n=7 each; p<0.01) in kidneys from sickle transgenic mice when compared to control transgenic knockout mice expressing human hemoglobin A (HbA). In addition, and as previously documented in other sickle mouse models, our sickle mice had increased circulating TNFα by ELISA as compared to HbA mice (p<0.05, n=6–10 of each mouse type). In conclusion, increases in ERAP1 levels represent a novel target in SCD that is likely secondary to hypoxia and activated endothelial cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling Melanoma in 3D uGel™‐based Artificial Tumor Tissues Demonstrate Initiation, Progression and Metastatic Processes In‐vitro
Authors: Lyden Timothy , Martin Mike
Published: June 2021

In a continuing collaboration between our laboratory, the UWRF Tissue and Cellular Innovation Center, and BRTI Lifesciences, LLC we have been evaluating the application of a commercial hydrogel as a matrix for the development and study of several artificial tumor tissues. Here we report on the application of uGel™, a recently developed chitosan/hyaluronate precast matrix to generate 3D melanoma artificial tissues. In this work, we coupled uGel™ matrix with mouse melanoma cell lines B16F10 and B16F1 to generate and study 3D tumor tissues in‐vitro. As we have previously reported with another earlier and related hydrogel, Cell‐Mate™, both of these cells lines successfully produced extensive and long‐lived tumor tissues. In addition, in this series of studies with uGel™ it was possible to model not just the end stages of tumor progression and metastasis, but also the early stages of tumor establishment and local invasion of the matrix in patterns which clearly reflect early tumor development in vivo. As we had seen before, the final stages of tumor development in melanoma includes the generation of two distinct populations of cells, one pigmented and one not. Additionally these two populations also correlate tightly with differential patterns of tissue formation ranging from highly branched and pigmented to completely rounded, smooth and lacking virtually any pigment. Although the branching features are clearly associated with local invasion, our work suggests that the pale, rounded features may actually represent the metastatic stage for these tumors. Interestingly, uGel™ based melanoma developed and progressed to full‐blown tumor masses within a three week period while in our earlier work with Cell‐Mate™ constructs fully developed tumors required from 2–3 months of long‐term culture to reach the same stage. Although the two materials are biochemically identical, they do differ in mechanical properties at the time of loading which may play a role in the significant differences observed in terms of developmental timeline. This study further support our view that small‐scale and long‐term 3D artificial tissues are an effective tool to better understand the biology of cancer and to study it's progression in‐vitro.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling Tumor Development, Progression and Metastasis in 3D Artificial Tissues: Characterization of Structure and Labeling Patterns in Breast Cancer Hanging‐drop Spheroid Cultures
Authors: Klinkhammer Katie , Leopold Marissa , Lyden Timothy
Published: June 2021

In this report our laboratory presents the results of a series of studies being conducted to explore the application of 3D artificial tumor tissues as in‐vitro models of breast cancer development, progression and metastasis. In this case, we have employed MCF‐7 breast ductal adenocarcinoma cells and 3D Biomatrix hanging drop plates to evaluate these tumor properties within the context of staged tumor spheroids. A consistent observation of ours in nearly a decade of modeling breast cancer using various natural 3D scaffolds and hydrogel matrices has been that solid tumor models grown in long‐term culture predictably begin to shed both single cells and cell clusters in the form of small groupings of cells as well as progressively larger spheroids. Although we have shown that at least some of these structures have properties consistent with metastasis, including attachment and invasion of target tissues, spontaneously generated versions of cancer clusters also present as pleiomorphic structures and in relatively small numbers which complicates their study. Therefore, we sought to use a system that generates identical clusters and spheres from specific numbers of suspended single cells. The hanging drop system we employ comes in a 96 well format that allows us to generate literally as many spheres as we need for each study. Furthermore, these spheres can be grown out to match any of the sizes commonly found in our tumor models thereby allowing us to study each stage for structure, cell populations and markers. In addition these spheres are also assisting us in systematically studying the process of cancer cell cluster attachment and invasion and to test our working hypothesis that this is controlled through activation of EMT‐like responses through CD44 binding of hyaluronate on the target tissue. Using direct labeling of intact spheres coupled to embedding and sectioning followed by immunolabeling, these studies are demonstrating that over a period of 144 hours our spheroid cultures progress from simple clusters of a few cells to aggregated spheres containing thousands of cells and measuring up to ~600um in diameter. By this later stage, many spheres actually begin to generate their own smaller spheres and clusters as well. In our view this reflects the concept that past 48 hours in culture these spheres transition into being actual tumor organoids themselves. This is supported by development of a complex CD24+ internal structure distinct from an outer shell of CD44+ cells. These studies are ongoing and support our view that 3D artificial tumors are effective tools to study cancer biology in‐vitro.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing a method of co‐incubating murine adipocytes and macrophages for evaluating cellular communication
Authors: Beck Jordan , Caldari‐Torres Cristina
Published: June 2021

Adipose tissue, an organ containing both adipocytes and adipose tissue macrophages (ATMs), secretes cytokines known as adipokines. Of these two cell types, ATMs have a larger role in the production and release of proinflammatory adipokines. The objective of this research was to develop a system of co‐incubating adipocytes and macrophages that allowed us to quantitatively measure the cross‐talk between these two cell types. Nitric Oxide (NO) and interleukin‐6 (IL‐6) concentrations were quantified to determine macrophage activation and production of a proinflammatory adipokine, respectively. Leptin concentrations were used as a measure of adipocyte‐specific adipokine production. The cell lines used were RAW 264.7 (murine macrophages) and 3T3‐L1 (murine fibroblasts). 3T3‐L1 fibroblasts were differentiated into adipocytes, which was confirmed by the presence of lipid droplets. Transwell plates with permeable inserts were used to co‐incubate the macrophages and adipocytes; the confluent adipocytes were maintained in the 12‐well plate (lower compartment) and the macrophages were added to the transwell insert (upper compartment). Macrophages were stimulated with 0.01 mg/ml of LPS either before or after the addition of the the macrophages to the insert. The treatments were: 3T3+media, 3T3+media+LPS, 3T3+unstimulatedRAW, 3T3+RAW+LPS, 3T3+stimulatedRAW (in fresh media), and 3T3+stimulatedRAW (in conditioned media). There was no difference in IL‐6 concentration measured in the upper compartment of the transwell insert vs the lower compartment, while measured NO levels were higher in the upper compartment and leptin levels were higher in the lower compartment (p<0.001, matched pairs t‐test). The 3T3+RAW+LPS, 3T3+stimulatedRAW (in fresh media) and 3T3+stimulatedRAW (in conditioned media) co‐incubation protocols resulted in the highest concentration of IL‐6 (p=0.0006). The 3T3+RAW+LPS and 3T3+stimulatedRAW (in conditioned media) co‐incubation methods yielded the highest concentrations of NO in both the upper and bottom compartments of the plate (p<0.001). There was no difference in leptin concentrations due to co‐incubation protocol. There was a positive relationship between NO and IL‐6 in both the top and bottom compartments (p<0.001, linear regression) and between NO and leptin in the top (p=0.03) but not in the bottom compartment. There was no significant relationship between IL‐6 and leptin concentrations in either the top or bottom compartments. For future experiments, our lab will utilize the co‐incubation protocols that resulted in the highest production of NO and IL‐6: 3T3+RAW+LPS and 3T3+stimulatedRAW (in conditioned media).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Expression g‐H2AX and RAGE in the Placenta of Gestational Diabetes Mellitus (GDM), Preterm Labor (PTL) and Preeclampsia Patients
Authors: Tsai K YF , Hirschi K M , Davis T , Llavina S , Knowlton M N , Bennet A , Reynolds P R , Arroyo J A
Published: June 2021

The integrity of the genome is fundamental for human health. Defective DNA repair response (DRR) is associated with several diseases, including pulmonary fibrosis, neurodegeneration, and in diseased placenta. The most destructive form of genomic damage is known to be DNA double strand breaks (DNA‐DSBs), in which both strands of DNA are ruptured. g‐H2AX is a component of the histone octomer and is phosphorylated in response to DNA‐DSBs. The receptor for advanced glycation end‐products (RAGE) is a multi‐ligand receptor primarily expressed on cell membranes, where it functions as a progression factor in inflammatory signaling; however, the nuclear isoform of RAGE (nRAGE) has been described as having a crucial role in DNA‐DSBs repair. The purpose of this study was to identify placental nRAGE and g‐H2AXexpression during normal gestation (Control), gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR), preterm delivery (PTL) and preeclampsia (PE). Immunohistochemistry was used to determine localization of both molecules present in human placenta. Immunoblot was further used to quantify nRAGE andg‐H2AX production. Assessing DNA degradation was used to confirm placental DNA damage during pregnancy complications. Relative to controls we observed: 1) Increased staining for of RAGE and g‐H2AX in PTL and PE placentas; 2) increased placental nRAGE protein in the PTL and PE placentas; 3) increased placental g‐H2AX protein in both PTL and PE conditions; and 4) increased DNA damage in the diseased placenta. These research suggest a potential role for nRAGE during DNA‐DSB detection and repair in human PTL and PE placentas. These results may provide insight into the physiological relevance of these molecules, and if so, their modification during gestation may help alleviate placental disease.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysophosphatidylcholine promotes trained immunity of human aortic endothelial cells via histone 3 lysine 14 acetylation‐dependent and independent gene modulation
Authors: Sun Yu , Lu Yifan , Yang William , Yu Daohai , Saaoud Fatma , Shao Ying , Cueto Ramon , Fu Hangfei , Wang Hong , Yang Xiaofeng
Published: June 2021

We previously reported that a conditional danger associated molecular pattern (conditional DAMP), pro‐atherogenic lipid lysophosphatidylcholine (lysoPC) induces human aortic endothelial cell (HAEC) activation via histone 3 lysine 14 acetylation (H3K14Ac); and HAEC activation plays important role in vascular inflammation and atherosclerotic cardiovascular diseases. However, the mechanisms underlying how lysoPC induced HAEC activation via histone acetylation remain unknown. To examine this issue, we carried out RNA‐Seq of lysoPC‐treated HAEC; and CHIP‐Seq using antibody to H3K14Ac in lysoPC‐treated HAEC. By analyzing the RNA‐Seq and CHIP‐Seq data, we found out the following results: 1) lysoPC modulates the expressions of 9 out of 31 histone acetyltransferases (HATs) with 6 increased and 3 decreased; and three out of the 6 increased HATs are reported to mediate H3K14Ac; 2) among three Acetyl‐CoA generating enzymes, lysoPC upregulates ACS2 (nucleus) and ACL (cytosol), and decreases ACS1 (peroxisome); 3) lysoPC modulates 5 out of 9 glycolytic enzymes with 3 increased and 2 decreased; and increases 6 out of 7 mevalonate pathway enzymes; 4) lysoPC increases the expressions of both T cell costimulation ligands and receptors; 5) lysoPC‐induced H3K14Ac increases the frequencies of downstream chromatin long range interactions (CLRIs); and lysoPC potentially‐induced other histone acetylations prefer to modulate upstream CLRIs. Our findings have demonstrated that lysoPC promotes HAEC activation and gene modulations via increasing acetylation of H3K14 and potential other histone lysines, and thereby frequencies of chromatin long‐range gene interactions, which significantly enhance newly characterized trained immunity features of HAEC. Our results provide novel therapeutic targets for controlling endothelial cell activation, vascular inflammation, and cardiovascular diseases.

Support or Funding Information

This work was supported by NIH grant to Drs. XF. Yang, H. Wang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Expression and Role of Epithelial‐Derived Gasdermin‐B (GSDMB) in the Context of Chronic Intestinal Inflammation
Authors: Rana Nitish , Pizarro Theresa T
Published: June 2021

Inflammatory bowel disease (IBD) represents a group of disorders that results in chronic, uncontrollable inflammation of the gastrointestinal (GI) tract. Despite recent advances, the precise etiology of the two major forms of IBD, Crohn's disease and ulcerative colitis, remains elusive. Gasdermin‐B (GSDMB), a novel molecule belonging to the Gasdermin family of proteins, has gained recent interest for its potential role in the pathogenesis and progression of chronic inflammatory disorders, particularly those that affect mucosal surfaces. Importantly, single nucleotide polymorphisms (SNPs) within the gene encoding for GSDMB have been reportedly associated with increasing the incidence of both asthma and IBD. Here, we aim to examine the aberrant expression and function of epithelial‐derived GSDMB towards the development and progression of chronic intestinal inflammation, such that observed in IBD. Using primary biopsy specimens derived from IBD patients, we confirmed the localization of GSDMB to primarily intestinal epithelial cells (IECs), both in protein and mRNA forms, via western‐blot techniques and immunohistochemistry staining. Moreover, preliminary evidence suggests an inflammasome dependent regulation of GSDMB (similar to that of the closely related GSDMD protein), via stimulation of IEC lines using lipopolysaccharides (LPS) and nigericin treatments to increase GSDMB mRNA expression. Due to the pore‐forming ability of the Gasdermin family of proteins, we suggest increased GSDMB expression may act as a membrane‐bound protein to facilitate the disruption of mucosal barrier function (i.e. restitution and repair), and act as conduit for release of inflammatory cytokines during the progression of IBD. Taken together, this data confirms an increased expression of epithelial‐derived GSDMB in the setting of chronic intestinal inflammation, providing indication for the role of GSDMB towards the development of inflammation within the gut mucosa during IBD.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelium‐Specific Acid Ceramidase Gene Deletion Enhances Exosome Secretion and Release of NLRP3 Inflammasome Products during Hyperglycemia
Authors: Yuan Xinxu , Bhat Owais M. , Lohner Hannah , Li Pin‐Lan
Published: June 2021

Exosomes have been demonstrated to be one of the mechanisms mediating the release of a variety of intracellular signaling molecules to mediate cell‐to‐cell communication. However, it remains unknown whether exosomes mediate the release of Nod‐like receptor pyrin domain 3 (NLRP3) inflammasome products such as IL‐1β from endothelial cells and how exosome‐mediated‐inflammatory signaling is regulated. The present study hypothesized that lysosomal acid ceramidase (AC) determines the fate of the multivesicular body (MVB) to control the exosome‐mediated release of NLRP3 inflammasome products during hyperglycemia. Using streptozotocin (STZ)‐induced mouse model of diabetes mellitus (DM), we found that endothelium‐specific AC gene knock‐out mice (Asah1fl/fl/ECcre) had significantly enhanced formation and activation of NLRP3 inflammasomes including IL‐1β production in the coronary arterial endothelium. These mice also had increased thickening of the coronary arterial wall and reduced expression of tight junction protein (ZO‐1 and ZO‐2) compared to their wild‐type (WT/WT) littermates. Interestingly, we observed that the expression of exosome markers such as CD63 and alkaline phosphatase (ALP) was also augmented in STZ‐treated Asah1fl/fl/ECcre mice compared to WT/WT mice, which was accompanied by an increased IL‐1β release of exosomes into the blood. In the primary cultures of coronary endothelial cells (CECs) from WT/WT and Asah1fl/fl/ECcre mice, we demonstrated that the AC deficiency markedly enhanced the formation and activation of the NLRP3 inflammasomes, but significantly down‐regulated tight junction proteins when CECs were exposed to high levels of glucose. The CECs from Asah1fl/fl/ECcre mice also had decreased MVBs and increased exosome release in response to high glucose stimulation and these exosomes contained increased IL‐1β. These results from our in vivo and in vitro experiments suggest that AC importantly controls exosome secretion and release of NLRP3 inflammasome products in CECs and its deficiency enhances this exosome‐mediated release of NLRP3 inflammasome products to activate coronary arterial inflammatory response during hyperglycemia.

Support or Funding Information

HL057244, HL075316, and DK120491

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sickle cell disease induced hepatopathophysiology involves reduced bile acid synthesis and transport
Authors: Pradhan‐Sundd Tirthadipa , Vats Ravi , Singh Sucha , Koshar Karis , Tutuncuoglu Egemen , Nejak‐Bowen Kari , Sundd Prithu , Monga Satdarshan P
Published: June 2021

Sickle cell disease (SCD) is an autosomal‐recessive‐genetic disorder that affects millions of people worldwide. Although hepatic crisis affects 10–40% of hospitalized SCD patients and can progress to fatal liver failure, the current treatment is primarily supportive and the molecular pathophysiology remains largely unknown. We found that transgenic, humanized SCD mice developed liver injury with age, manifested by progressive hepatomegaly, hyperbilirubinemia, iron accumulation, dyslipidemia, fibrosis, increased inflammation and necrosis. Remarkably, real‐time in‐vivo imaging of liver in live mice revealed presence of sinusoidal ischemia, impairment of bile‐secretion, and accumulation of bile‐acids in SCD but not control mice. Interestingly, impaired bile secretion was associated with the loss of apical bile‐transporters and bile‐acid converting enzymes as well as gain of efflux bile‐transporters. These findings are the first to identify that impaired bile acid synthesis and misexpression of bile transporters in SCD, contribute to liver injury by promoting intrahepatic bile accumulation and impaired bile secretion into bile canaliculi. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Post‐Natal Growth and Liver Immune Homeostasis are Post‐Transcriptionally Regulated by Tristetraprolin Family of RNA Binding Proteins
Authors: Patial Sonika , Blackshear Perry
Published: June 2021

Tristetraprolin family of RNA binding proteins, including tristetraprolin (TTP), zinc finger protein 36 like 1 (ZFP36L1), and zinc finger protein 36 like 2 (ZFP36L2) regulate mRNA levels by binding to AU‐rich elements on the 3′ untranslated regions of specific mRNAs and enhancing their turnover. While the three proteins behave similarly under biochemical conditions, they appear to have diverse physiological functions. This is apparent from the global deletion of the three proteins in mice. While the global loss of TTP results in systemic inflammatory syndrome, global loss of ZFP36L1 is embryonic lethal, and the global loss of ZFP36L2 results in early post‐natal mortality. Here, we sought to understand the physiological functions of the three proteins in liver by conditional liver specific deletion using Cre‐loxP technology. While the independent deletion of the three proteins did not result in any aberrant phenotype, simultaneous deletion resulted in post‐natal growth defect, chronic active portal hepatitis, and bile duct hyperplasia. Transcriptomic analysis showed small numbers of gene expression changes in the liver of ZFP36L1 knockout, no changes in the livers of TTP and ZFP36L2 knockout, and many changes in the liver of mice deleted of the three proteins simultaneously. The changes included perturbation of genes that regulate post‐natal growth and several inflammatory mediators, some of which are novel potential liver‐specific targets of TTP family proteins. Interestingly, one allele of either ZFP36L1 or ZFP36L2 was sufficient to protect the mice from developing this phenotype. However, one allele of TTP was only partially protective. In summary, our results suggest that post‐transcriptional regulation of gene expression plays an important role in post‐natal growth and liver immune homeostasis and that TTP family proteins regulate these physiological functions in both specific and redundant manner.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improved Method to Capture a Broader Array of High Density Lipoprotein Particles Including Those of Intestinal Origin
Authors: Kang Jea Woo , Rhodes Christopher H. , Houts Hannah , Zivkovic Angela M.
Published: June 2021

The isolation of high density lipoprotein (HDL) particles by density‐based methods using a density range of 1.063–1.21 g/mL is considered to be the gold standard. However, it is known that several HDL subclasses, including discoidal/nascent HDL particles, as well as ApoE‐containing, and intestinally derived ApoA‐IV‐containing HDL particles, are found at a higher density of up to 1.25 g/mL. In addition, using density‐based approaches alone it is not possible to purify HDL particles from a range of other particles such as extracellular vesicles, which overlap in density but not size. In this study, a novel method was optimized to isolate a broader array of HDL particles in a purified fraction that minimizes contamination by extracellular vesicles. The method utilizes density‐based sequential flotation ultracentrifugation followed by size exclusion chromatography with an FPLC system to isolate HDL fractions of different sizes, which were then analyzed for their cholesterol efflux capacity and protein content. The results demonstrate a higher yield of HDL and an increased level of HDL from earlier fractions using the 1.25 density cut‐off compared to the 1.21 density cut‐off. The cholesterol efflux capacity of early and late eluting fractions from the 1.25 density cut‐off method is increased, suggesting that both smaller and larger higher‐density particles, both with increased efflux capacity, are being isolated with the new method. Gel electrophoresis and Western Blot results from these fractions indicate enrichment of ApoE as well as ApoA‐IV in HDL fractions collected using the 1.25 density cut‐off method, particularly from plasma samples collected in the postprandial vs. the fasted state. In conclusion, we demonstrate that the use of the optimized HDL isolation method yields a broader array of HDL particles, which may be lost when the 1.21 density cut‐off is used.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microvessel Stenosis and Density Analysis of Post Mortem WMH Detected Using Ultra High Field MRI in Aging, Cerebrovascular and Alzheimer Disease
Authors: Roseborough Austyn , Langdon Kris , Hammond Robert , Pasternak Stephen , Khan Ali , Whitehead Shawn
Published: June 2021

Cerebral small vessel disease (SVD) affects the microvasculature of the brain, increasing in prevalence with age and associating with cognitive decline and dementia. SVD can be visualized on MR imaging by markers such as hyperintensities within the white matter (WMH). Periventricular WMH within the frontal lobe specifically may have a negative effect on executive functions. Executive dysfunction represents a set of cognitive changes, including processing speed and attention, that can precede memory impairment in Alzheimer disease. The etiology and development of cerebral small vessel disease, including WMH, and it's relation to executive dysfunction is not well understood. Previous work has characterized the presence of stenosis of the arterioles, but little work has focused on the venules. In this study we sought to characterize alterations in microvascular stenosis and density including small and medium venules and arterioles within the frontal white matter. Post mortem 7‐Tesla MR imaging of formalin‐fixed coronal brain sections was performed on 20 brains with a neuropathological diagnosis of normal, cerebrovascular disease or Alzheimer disease. We calculated the percent stenosis within small, medium and large venules and arterioles within both the periventricular and subcortical white matter. Stenosis of the small (<50 um diameter) arterioles and venules within the periventricular white matter specifically was associated with increased severity of periventricular WMH. Small vein and artery stenosis was also associated with the presence of periventricular infarction identified on T1‐weighted imaging. This highlights the utility of multiple sequences when interpreting MRI findings within the white matter. Further study on markers of small vessel disease that affect executive functioning, such as frontal WMH, can help elucidate mechanisms that lead to cognitive decline prior to the onset of memory impairments and dementia.

Support or Funding Information

Canada First Research Excellence Fund to BrainsCAN

Canadian Institutes of Health Research

Canadian Consortium on Neurodegeneration in Aging

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pericytes Contribute to Retinal Vascular Cell Death in Diabetes: Role of Ephrin‐B2 Signaling
Authors: Coucha Maha M , Barrett Amy C , Abdelsaid Mohammed A
Published: June 2021

Diabetes‐induced retinal vascular cell death aggravates diabetic retinopathy to proliferative stage and blindness. Pericytes (PCs) share the basement membrane with endothelial cells (EC) in retinal capillaries and play a crucial role in capillaries survival, stability, and angiogenesis. Ephrin‐B2 is a tyrosine kinase that has an essential role in PC/EC communication. The role of Ephrin‐B2 signaling in diabetic retinopathy is under investigation. In the present study, we hypothesized that diabetes alters Ephrin‐B2 signaling in pericytes that increase inflammation and retinal vascular cell death.

Methods

Type‐II diabetes was induced in male Wistar rats using high‐fat diet and streptozotocin. In vivo, selective reduction of Ephrin‐B2 expression in the retinal PC was achieved using the intraocular injection of AAV‐virus with NG2‐promoter that expresses EphrinB2 shRNA. Retinal trypsin‐digest was used to assess acellular capillary. Human retinal microvascular (HRP) were grown in glucose 25 mM/palmitate 200 uM (HG/Pal) to mimic diabetic conditions. Ephrin‐B2 was silenced in HRPs using siRNA. Cell migration was assessed using a scratch assay. Ephrin‐B2 expression, inflammatory (TNF‐alpha, JNK and IL‐1 beta) and apoptotic (PARP, Caspase‐3) markers were assessed via Western blot and immunohistochemistry.

Results

Diabetes increased Ephrin‐B2 expression in the retinal perivascular areas and pericytes that was confirmed with Western blot analysis. HRP treated with diabetic conditions showed a significant increase in Ephrin‐B2 expression that was associated with increased HPR migration. Silencing Ephrin‐B2 expression significantly reduced PC migration. Increased Ephrin‐B2 expression in pericytes in diabetes was accompanied with an increase in inflammatory markers TNF‐alpha, JNK and IL‐1 beta. In parallel diabetes increases PARP and caspase‐3 cleavage in diabetic retina. Silencing Ephrin‐B2 in retinal PCs decreased retinal vascular inflammation and apoptosis that was in parallel with the decrease in the number of acellular capillaries in retinas of diabetic rats.

Conclusion

Our results provide novel evidence that diabetes increases Ephrin‐B2 signaling in retinal pericytes that increase retinal vascular inflammation and death. These results identify pericytes‐Ephrin‐B2 signaling as a novel therapeutic target for diabetic retinopathy.

Support or Funding Information

AHA, 16SDG30270013 to MA.

SUSOP Excellence in Pharmacy Research award to MC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective effects of different bile acids against pathological neovascularization
Authors: Thounaojam Menaka C. , Gutsaeva Diana R. , Martin Pamela M. , Bartoli Manuela , Jadeja Ravirajsinh
Published: June 2021

Emerging evidence suggest that the secondary bile acid ursodeoxycholic acid (UDCA) and its taurine (TUDCA) conjugates, possess neuroprotective and vasculoprotective properties and may exert beneficial effects in a number of neovascular diseases including ischemic retinopathy such as retinopathy of prematurity (ROP). However, the direct activity of UDCA or TUDCA in the treatment of retinal neovascularization has not been previously investigated. Herein, we studied the effects of different bile acids on in vitro angiogenesis in human retinal endothelial cells (HuREC) and retinal vascularization in vivo using the mouse model of oxygen induced retinopathy (OIR), an experimental model of ROP. HuREC were treated with 20 ng/ml of VEGF and/or different doses (10–500 μM) of UDCA or TUDCA for different times. Angiogenic responses were evaluated using tube formation assay. Treatment of HuREC with VEGF significantly induced time dependent tube formation. UDCA dose‐dependently halted VEGF‐induced tube formation, whereas TUDCA had no effect. For the in vivo studies, mice were subjected to OIR and treated with UDCA and TUDCA (administered i.p.) from days P12–P17 (the neovascular stages). Isolectin B4 staining on flat mounted retinal preparations was performed at P17 to evaluate retinal vessel growth and distribution. The results of this analysis revealed that UDCA in the OIR mice halted pathological neovascularization, but did not alter normal vascularization. On the contrary, treatment of the mice with TUDCA failed to block retinal neovascularization, thus confirming the in vitro data. In summary, here we have shown that the bile acids UCDA and TUCDA possess differential ability to modulate VEGF‐mediated angiogenesis effects on HuREC and on retinal pathological neovascularization in vivo. The results of studies support the hypothesis that UDCA could be a potential therapeutic option for ROP as it exerts protection even by systemic administration and does not alter normal progression of vessel formation.

Support or Funding Information

This study is supported by R03HD097660‐01.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Simultaneous knockdown of TLR2 and TLR4 after focal cerebral ischemia facilitates the post‐stroke neurological recovery in rats
Authors: Nalamolu Koteswara Rao , Jorgenson Laura C , Grudzien Natalia A , Choudry Mouneeb M , LaHood Lukas D , Johnson Kendrick D , Klopfenstein Jeffrey D , Pinson David M , Wang David Z , Veeravalli Krishna Kumar
Published: June 2021

Ischemic stroke is the primary reason for serious long‐term disability. Our recent studies demonstrated that the suppression of toll‐like receptors (TLRs) 2 and 4 alone or in combination attenuated the post‐ischemic inflammation and brain damage. The purpose of this study is to investigate the effect of the simultaneous knockdown of both TLR2 and TLR4 on post‐stroke neurological recovery in young adult male rats. Transient focal cerebral ischemia was induced to male Sprague‐Dawley rats by middle cerebral artery occlusion (MCAO) procedure. A silicone rubber‐coated monofilament suture was introduced into the internal carotid artery via the external and common carotid arteries and pushed until it reaches the origin of the middle cerebral artery followed by removal of the monofilament after 2 hours to initiate reperfusion. Appropriate cohorts of rats were treated with a nanoparticle formulations of TLR2shRNA (T2sh) and TLR4shRNA (T4sh) expressing plasmids (1mg/kg each of T2sh and T4sh) or scrambled sequence inserted vector (SVsh; vehicle) expressing plasmids (2 mg/kg) intravenously via tail vein immediately after reperfusion. The rats from both the cohorts were subjected to various standard neurological tests such as modified neurological severity scores (mNSS), modified adhesive removal (sticky‐tape), beam walking and accelerating rotarod performance before MCAO procedure and at days 1, 3, 5, 7, and 14 after reperfusion. The sensory and motor functions of vehicle treated stroke‐induced rats were markedly impaired when tested at 1d reperfusion followed by a gradual improvement over 14 days. Prevention of the post‐ischemic induction of TLR2 and TLR4 attenuated the degree of neurological impairment and facilitated the magnitude of functional recovery. Therefore, we conclude that the post‐stroke induction of TLR2 and TLR4 in rat ischemic brain contributes to neurological deficits.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aliskiren Effect on Kidney Renin‐Prorenin Stained Cells in a Rat Model for Fat Embolism
Authors: Khalafi Farnaz , Randall Zach , Leupold Brad , Silswal Neerupma , Monaghan Paula , Poisner Alan , Molteni Agostino
Published: June 2021

Introduction

The renin angiotensin system (RAS) is involved in the control of blood pressure through its action on the kidney and the treatment by the direct renin inhibitor Aliskiren (Ali) reduces it. We have previously shown in a rat model of fat embolism (FE) that triolein (T) injection leads to a severe inflammatory pulmonary reaction that causes fibrosis in lungs and Ali rescues it. We extended this study to the kidney by evaluating the renin/prorenin (R/P) presence in the kidney with and without Ali in a rat model of fat embolism.

Methods

Sprague Dawley rats (280–300 grams) were injected with T (0.2 ml IV, n=18) or saline (n=4) through tail vein. The T treated rats were divided into three groups of 6 rats each and one hour later injected with 0.2 ml saline, Ali 50mg/kg or Ali 100mg/kg while 4 controls received saline. After 48 hours, rats were euthanized, the kidneys were dissected out and fixed in 10 % formalin, sectioned and later immunostained for R/P specific antibody (from Abcam). Two pathologists unaware of the slide identity took 10 random images. R/P immunostained cells were quantified by using ImageJ program on medulla and cortical regions where the glomeruli were evident.

Results

In contrast to our previous results in the lungs of the same animals, the renal sections showed a minimal inflammatory damage without significant vasculitis and fibrosis. There are multiple cells cortical, tubular and glomerular showing the R/P cells immunostaining. It was evident by quantification of these cells by Image J that treatment with saline, T + saline, and T+ Ali resulted in an increase of R/P positive cells versus the saline + saline group but there was no statistically significant difference. However, in the glomeruli of the cortical region, R/P staining was mostly confined in the juxtaglomeruli of saline + saline and T+ saline groups and treatment with both doses of Ali reduced it and was statistically significant.

Conclusion

As previously reported for lungs and hearts, R/P positive cells were also present in kidneys. However in kidney, marked differences in several regions were seen after Ali administration. Furthermore, by reducing the R/P evaluation only to glomeruli, the study confirms that Ali pharmacological activity is interacting with the RAS system, and the drug has a potential use for the treatment of the respiratory distress syndrome consequent to the occurrence of fat embolism.

Support or Funding Information

Supported by Mary Katherine Geldmacher Research Foundation, St. Louis MO.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The lack of Galectin 3 modifies the evolution of the healing and post‐ myocardial infarct remodeling in mice
Authors: Betazza Maria Celeste , Estevez Florencia Fontana , Cassaglia Pablo , Wilensky Luciana , Cozzi Maria , Truant Sofia Noli , Miksztowicz Veronica , Fernandez Marisa , Naya Nadia Laura Martinez , Volberg Veronica , Berg Gabriela , Casanova Veronica , Morales Celina , Gonzalez German Esteban
Published: June 2021

Myocardial infarction (MI) is a dynamic process that leads to ventricular remodeling (VR) and heart failure (HF). Previous studies established that Galectin‐3 (Gal‐3), is increased during infarction and it is a prognostic marker of HF. Here, we aimed to study the effects of genetic mutation of Gal3 on macrophages infiltration, fibrosis and MMP‐2 as well as VR after MI in mice. Adult male C57 and Gal3KO mice were subjected to permanent coronary artery ligature and sham. After 1 week (w) post‐MI, echocardiography was performed. LV end‐diastolic diameter (LVEDD) was measured and ejection fraction (EF%) calculated. Then, animals were euthanized. Body (BW) and heart weight (HW), tibial length (TL) and lung wet weight (LW) were measured. Hearts were harvested, snap frozen and storage at −80°C. Infarct size and fibrosis in MI area were quantified in cardiac slices stained with Masson's Trichrome and Picrosirius Red. Myocyte cross sectional area (MCSA) was also measured in slices labeled with wheat germ agglutinin–rhodamine. At 1w post‐MI macrophages (F4/80+ cells) were quantified by flow cytometry and MMP‐2 activity was studied by zymography, both in the MI area.

Results

are expressed as X±SEM; * p<0.05 C57 vs Gal3KO. At 1w post‐MI, the pulmonary congestion assessed by the LW/BW ratio (mg/g) was 9 ± 0.4, 8 ± 0.5 and 9 ± 0.4 in C57 sham, Gal3 KO sham and C57 MI, respectively, but it was severely increased to 15 ± 1.2* in the Gal‐3 KO MI. The macrophage infiltration (%) was reduced from 5.6 ± 0.9 to 2.6 ± 0.4 and the fibrosis (%) in infarct zone was reduced from 30 ± 1.0 to 18 ± 0.4* in C57+MI and Gal3KO+MI respectively. At the same time, MMP2 activity increased from 1 ± 0.1 (C57+MI) to 2 ± 0.4*(Gal3KO+MI). The Infarct size (%) increased from 39 ± 5 to 67± 5* in C57+MI and Gal3KO+MI respectively. Moreover, EF (%) was reduced from 47 ± 2 to 38 ± 3*, while the LVEDD was increased from 4.4 ± 0.1 to 4.8 ± 0.2* in C57+MI and Gal3KO+MI respectively. Fibrosis at remote zone as well as the hypertrophy index evaluated through the HW/BW ratio (mg/g) were similar between groups. In summary, Gal‐3 is essential for the early wound healing and VR in mice. The lack of Gal‐3 reduces the infiltration of macrophages and fibrosis while increasing the MMP‐2 activity, the infarct size and expansion, LV dilation and dysfunction. New studies should focus on the role of Gal3 on the macrophage phenotype profiles, cytokine expression and chronic remodeling.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Ultrafine Nano‐zinc Particles on Cardiac Structure and Function in Myocardial Infarction Mice
Authors: Huang Yufan , Huo Yunlong
Published: June 2021

Objective

We aimed to illustrate the association between ultrafine nano zinc particles inhalation and cardiac structure and function in myocardial infarction (MI) mice.

Method

6‐week‐old ICR mice were divided into three groups randomly: sham group, MI group and MI exposure group, 12 mice in each group. We preformed LAD ligation operation in MI group and MI exposure group. After postoperative two weeks MI exposure mice were put into ventilation chamber which filled with 500ug/m3 ultrafine nano zinc particles for 6 hours per day, while MI group mice and sham group mice were cultivated in normal environment. After exposure 8 weeks, we use Vevo 2100 machine to acquire heart function measurements. Then we collected blood sample and killed mice and obtain heart samples that were used for immunohistochemistry. The apoptosis myocardium were measured by immunofluorescence. Elisa method was used to detect the catecholamine expression in plasma. The changes of collagen were measured by Masson trichrome staining.

Results

Compared with the sham group, the EF in MI exposure group and the MI group decreased significantly (p<0.05), and the decrease in MI exposure group was more obvious. The results of immunofluorescence showed that the number of apoptotic cells in MI exposure group increased significantly (p<0.05) and the myocardial cells exaggerated significantly. In addition, the norepinephrine in the peripheral blood of MI exposure group was increased significantly (p<0.05), in the same time the Marsson trichrome staining results showed that the collagen content of MI exposure group's myocytes increased significantly (p<0.05).

Conclusion

Nano‐zinc particles inhalation can aggravated ventricular remodeling and heart dysfunction and increased myocardial cell apoptosis after myocardial infarction in mice.

Support or Funding Information

Shenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.