Biology subjects

Weaver, V.

Publications and source records attributed to Weaver, V..

7 recordsLinked to original sources

bioRxiv2024

Assessing Heterogeneity in the N-Telopeptides of Type I Collagen by Mass Spectrometry

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.

biochemistry↗

bioRxiv2023

The microenvironment dictates glycocalyx construction and immune surveillance

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium. Key PointsO_LICulture medium dictates cellular mechanoresponse signatures in vitro C_LIO_LIEpithelial glycocalyx construction is mediated by Heat Shock Factor 1 (HSF1) C_LIO_LISialic acid topology dictates Natural Killer cell cytotoxicity C_LIO_LIPhysiological microenvironments reveal distinct glycobiology C_LI

cell biology↗

bioRxiv2022

Myeloid mechano-metabolic programming restricts anti-tumor immunity

Tumor progression is accompanied by fibrosis, which is associated with diminished anti-tumor immune infiltrate. Here, we demonstrate that tumor infiltrating myeloid cells respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a TGF-beta (TGF{beta})-directed, collagen biosynthesis program. A collateral effect of this programming is an untenable metabolic milieu for productive CD8 T cell anti-tumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline, and secrete ornithine that compromises CD8+ T cell function. Thus, a stiff and fibrotic TME may impede anti-tumor immunity not only by direct physical exclusion of CD8+ T cells, but also via secondary effects of a myeloid mechano-metabolic programming we identified that creates an inhospitable metabolic milieu for CD8+ T cells.

cancer biology↗

bioRxiv2022

Vitamin D and the ability to produce 1,25(OH)2D are critical for protection from viral infection of the lungs.

Vitamin D supplementation has been linked to improved outcomes from respiratory virus infection, and the COVID19 pandemic has renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of Vitamin D using animal models of pandemic H1N1 influenza and SARS-CoV-2 infection. In mice, dietary induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D-) wildtype (WT) and D+ and D-Cyp27B1 (Cyp) knockout (KO, cannot produce 1,25(OH)2D) mice were infected with pandemic H1N1. D- WT, D+ Cyp KO, and D- Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficient/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.

immunology↗

bioRxiv2022

Microenvironmental engineering improves the self-organization of patterned microvascular networks

The construction of three-dimensional (3D) microvascular networks with defined structures remains challenging. Emerging bioprinting strategies provide a means of patterning endothelial cells (ECs) into the geometry of 3D microvascular networks, but the microenvironmental cues necessary to promote their self-organization into cohesive and perfusable microvessels are unknown. To this end, we reconstituted microvessel formation in vitro by patterning thin lines of closely packed ECs fully embedded within a 3D extracellular matrix (ECM) and observed how different microenvironmental parameters influenced EC behaviors and their self-organization into microvessels. We found that the inclusion of fibrillar matrices, such as collagen I, into the ECM positively influenced cell condensation into extended geometries such as cords. We also identified the presence of a high molecular weight protein(s) in fetal bovine serum (FBS) that negatively influenced EC condensation. This component destabilized cord structure by promoting cell protrusions and destabilizing cell-cell adhesions. Endothelial cords cultured in the presence of fibrillar collagen and the absence of this protein activity were able to polarize, lumenize, incorporate mural cells, and support fluid flow. These optimized conditions allowed for the construction of branched and perfusable microvascular networks directly from patterned cells in as little as three days. These findings reveal important design principles for future microvascular engineering efforts based on bioprinting techniques.

bioengineering↗

bioRxiv2021

Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells have lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells altered expression of numerous other proteins implicated in metastatic progression. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels and decreased disease-free survival. These findings suggest that downregulation of lamin A/C may influence both biochemical and physical properties of the cell to promote breast cancer metastatic progression.

cancer biology↗

bioRxiv2019

RNAseq studies reveal distinct transcriptional response to vitamin A deficiency in small intestine versus colon, discovering novel VA-regulated genes

Vitamin A (VA) deficiency remains prevalent in resource limited countries, affecting over 250 million preschool aged children. Vitamin A deficiency is associated with reduced intestinal barrier function and increased risk of mortality due to mucosal infection. Using Citrobacter rodentium (C. rodentium) infection in mice as a model for diarrheal diseases in humans, previous reports showed reduced pathogen clearance and survival in vitamin A deficient (VAD) mice compared to their vitamin A sufficient (VAS) counterparts. ObjectivesTo characterize and compare the impact of preexisting VA deficiency on gene expression patterns in the small intestine (SI) and the colon, and to discover novel target genes in VA-related biological pathways. MethodsVAD mice were generated by feeding VAD diet to pregnant C57/BL6 dams and their post-weaning offspring. RNAseq were performed using the total mRNAs extracted from SI and colon. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression and co-expression patterns. ResultsDEGs compared between VAS and VAD groups detected 49 SI and 94 colon genes. By GO information, SI DEGs were significantly enriched in categories relevant to retinoid metabolic process, molecule binding, and immune function. Immunity related pathways, including "humoral immune response" and "complement activation" were positively associated with VA in SI. Three co-expression modules showed significant correlation with VA status in SI; these modules contained four known retinoic acid targets. In addition, other SI genes of interest (e.g. Mbl2, Cxcl14, and Nr0b2) in these modules were suggested as new candidate genes regulated by VA. Furthermore, our analysis showed that markers of two cell types in SI, mast cells and Tuft cells, were significantly altered by VA status. In colon, "cell division" was the only enriched category and was negatively associated with VA. Thus, comparison of co-expression modules between SI and colon indicated distinct networks under the regulation of dietary VA and suggest that preexisting VAD could have a significant impact on the host response to a variety of disease conditions.

physiology↗