Biology subjects

O'Hara-Smith, J. R.

Publications and source records attributed to O'Hara-Smith, J. R..

4 recordsLinked to original sources

bioRxiv2026

Active and Passive Mechanical Deficits Precede Spinal Curvature in a Zebrafish Model of Idiopathic Scoliosis

Idiopathic scoliosis is a common spinal disorder characterized by progressive three-dimensional curvature of unknown cause. Although biomechanical imbalance has long been proposed to contribute to scoliosis, the early physiological states that precede curvature onset remain poorly understood. Here, we investigated this problem using zebrafish uts2r3 mutants, which develop fully penetrant juvenile-onset spinal curvature following disruption of urotensin signaling. Transcriptomic analysis before curvature revealed altered expression of muscle-associated genes, suggesting that Uts2r3 influences axial muscle development or function. However, immunofluorescence, birefringence imaging, and quantitative analysis of myotome morphology showed that mutants lack overt muscle architectural defects or dystrophic pathology. By contrast, direct measurements of isolated larval trunks revealed pre-curvature biomechanical abnormalities: namely, uts2r3 mutants generated reduced active force following electrical stimulation while also exhibiting increased passive resistance to stretch. These findings identify urotensin signaling as a regulator of axial tissue biomechanics during growth and suggest that scoliosis-like curvature can arise from an early imbalance between active force generation and passive tissue stiffness. SignificanceSpinal curvature is common, but the biological events that cause the spine to bend during growth remain poorly understood. Animal models, especially zebrafish, make it possible to study these events before curvature begins. Zebrafish lacking urotensin signaling develop spinal curves that arise during juvenile growth, similar to idiopathic scoliosis in humans. Here, we demonstrate that zebrafish lacking the urotensin pathway receptor Uts2r3 develop an abnormal biomechanical state prior to curve onset. Their axial tissues generate less active force when contracting and, at the same time, show increased passive resistance to stretch--an unexpected combination that reveals a distinct pre-curvature biomechanical state. These findings suggest that spinal curvature can arise from an early imbalance in tissue mechanics during growth and identify urotensin signaling as a pathway that helps preserve spinal morphology through a biomechanical mechanism.

developmental biology↗

bioRxiv2025

Phased affinity-controlled delivery of vascular endothelial growth factor, fibroblast growth factor-2, and platelet derived growth factor enhances in vitro angiogenesis

Angiogenesis, the growth of vasculature from existing blood vessels, requires the coordinated secretion of multiple angiogenic growth factors that each stimulate the cellular recruitment, patterning, and morphogenesis inherent to vascular network formation. Among these secreted factors, vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet derived growth factor (PDGF) amplify key stages of angiogenesis. Disruptions in their secretion have been implicated in poor vascular network formation. Current methods for exploring variations in the phased presentation of multiple different proteins are limited, which has restricted our ability to explore the effect of growth factor timing on angiogenesis. To address this knowledge gap, we developed affibodies, which are alpha-helical binding proteins, to phase the release of VEGF-165, FGF-2, and PDGF-BB from a single delivery vehicle via specific protein-affibody affinity interactions. We used yeast surface display to engineer three VEGF-, three FGF-2-, and two PDGF-specific affibodies with a wide range of affinities for their target proteins spanning dissociation constants of 3.08 {+/-} 0.21 nM to 4550 {+/-} 590 nM. We demonstrated that the cumulative release of VEGF and FGF-2 is inversely correlated with the strength of the protein-affibody affinity interaction and that hydrogels containing multiple protein-specific affibodies can control the release of VEGF, FGF-2, and PDGF, largely in accordance with the strength of the affinity interactions. Using a rat-derived intact microvascular fragment model of in vitro angiogenesis, we revealed that sequential delivery of soluble VEGF, followed by FGF-2, and then PDGF enhances vascular network length by 2.8-fold and branching by 4.1-fold compared to untreated MVFs. We then designed an affibody-conjugated polyethylene glycol maleimide (PEG-MAL) hydrogel to mimic this sequence of protein delivery, resulting in a 3.0-fold increase in vascular network length and a 2.3-fold increase in vascular branching compared to all other hydrogel compositions and the sequential delivery of soluble growth factors. Changing temporal growth factor presentation with affibody-conjugated hydrogels altered the expression of key angiogenic genes involved in vessel stabilization and destabilization and matrix remodeling. Perivascular coverage measured by the colocalization of lectin and alpha smooth muscle actin staining was similar between all treatment groups, suggesting pericyte recruitment to stabilize expanded vascular networks created by the soluble and affibody-mediated delivery of the optimal sequence of proteins. Overall, this work establishes a new biomaterial platform for modulating the timing of growth factor delivery, enabling the exploration of how temporal variations in protein secretion impact regeneration and development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/662647v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@471657org.highwire.dtl.DTLVardef@10bc491org.highwire.dtl.DTLVardef@1961f9dorg.highwire.dtl.DTLVardef@f5b8ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Applying computational protein design to engineer affibodies for affinity-controlled delivery of vascular endothelial growth factor and platelet-derived growth factor

Effective angiogenesis requires the coordinated presentation of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which play competing roles in stimulating vascular outgrowth and stabilization. Dysregulation in VEGF and PDGF secretion are implicated in abnormal vascular structures, poor vessel stability, and inadequate tissue repair. Current biomaterial delivery vehicles for these proteins have a limited ability to precisely control the kinetics of protein release, preventing systematic exploration of their temporal effects. Here, we combined yeast surface display and computational protein design to engineer eight VEGF-specific and PDGF-specific protein binders called affibodies with a broad range of affinities (dissociation constants = 2.23-9260 nM) for affinity-controlled protein release. Starting from one VEGF- and one PDGF-specific affibody discovered from yeast surface display, computational modeling was used to select disruptive mutations to create VEGF-specific affibodies with lower affinities for VEGF and expand the affinity range of PDGF-specific affibodies. In both cases, the specificity of the affibodies for their target protein was either maintained or enhanced. Soluble protein-specific affibodies modulated protein bioactivity as evidenced by changes in VEGF-induced endothelial cell proliferation and luminescent output of a PDGF-responsive fibroblast cell line. Affibody-conjugated polyethylene glycol maleimide (PEG-mal) hydrogels sustained VEGF and PDGF release compared to hydrogels without affibodies, enabling tunability of protein release over 7 days. VEGF and PDGF released from affibody-conjugated hydrogels exhibited higher bioactivity compared to proteins released from hydrogels without affibodies, highlighting the potential of these engineered affinity interactions to prolong protein bioactivity. This work underscores the power of computational protein design to enhance biomaterial functionality, creating a platform for tunable protein delivery to support angiogenesis and tissue repair.

bioengineering↗

bioRxiv2022

Urotensin II-Related Peptides, Urp1 and Urp2, Control Zebrafish Spine Morphology

The spine provides structure and support to the body, yet how it develops its characteristic morphology as the organism grows is little understood. This is underscored by the commonality of conditions in which the spine curves abnormally such as scoliosis, kyphosis and lordosis. Understanding the origin of such spinal curves has been challenging in part due to the lack of appropriate animal models. Recently, zebrafish have emerged as promising tools with which to understand the origin of spinal curves. Using zebrafish, we demonstrate that the Urotensin II-related peptides (URPs), Urp1 and Urp2, are essential for maintaining spine morphology. Urp1 and Urp2 are 10-amino acid cyclic peptides expressed by neurons lining the central canal of the spinal cord. Upon combined genetic loss of Urp1 and Urp2, adolescent-onset planar curves manifested in the caudal region of the spine, akin to a lordosis-like condition. Highly similar curves were caused by mutation of Uts2r3, an URP receptor. Quantitative comparisons revealed that Urotensin-associated curves were distinct from other zebrafish spinal curve mutants that more closely reflected idiopathic scoliosis or kyphosis. Last, we found that the Reissner fiber, a proteinaceous thread that sits in the central canal and has been implicated in the control of spine morphology, breaks down prior to curve formation in an idiopathic scoliosis model but was unperturbed by loss of Uts2r3. This suggests a Reissner fiber-independent mechanism of curvature in Urotensin-deficient mutants. Overall, our results show that Urp1 and Urp2 control zebrafish spine morphology and establish new animal models of lordosis-like curves.

developmental biology↗