Biology subjects

Madine, J.

Publications and source records attributed to Madine, J..

6 recordsLinked to original sources

bioRxiv2026

Medin-Induced Pro-inflammatory and Prothrombotic Activation of Coronary Artery Endothelial Cells: A Potential Novel Mediator Linking Aging and Atherosclerosis

BackgroundAge is the most important risk factor for coronary artery disease (CAD) independent of traditional risk factors. Aging induces classic pro-inflammatory and prothrombotic vascular phenotypic changes whose molecular mediators remain poorly understood. Medin is a common cleavage product protein that accumulates in vasculature with aging and shown to cause endothelial dysfunction. Its role in CAD is unknown. The study aimed to evaluate the effects of medin on human coronary artery endothelial cell (HCAEC) pro-inflammatory and prothrombotic activation and establish the relationship between medin and coronary atherosclerosis in human decedents. MethodsHCAECs were exposed to physiologic dose of medin (5 {micro}M) for 20 hours and ribonucleic acid sequencing (RNAseq) with signaling pathway analyses and reverse transcription polymerase chain reaction of select pro-inflammatory and prothrombotic genes performed. Corresponding protein expression was measured by Western blot or enzyme linked immunosorbent assay in HCAECs exposed to medin (5 {micro}M) without or with nuclear factor-{kappa}B (NF{kappa}B) inhibitor RO106-9920 (10 {micro}M). Coronary arteries from 40 deceased individuals underwent immunohistochemistry and medin and plaque burden were quantified and their relationship evaluated. ResultsRNAseq showed predominant pro-inflammatory gene expression changes induced by medin. HCAECs treated with medin showed increased phosphorylated NF{kappa}B, elevated protein expression of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and plasminogen activator inhibitor (PAI)-1 and reduced protein expression of thrombomodulin; these changes were reversed by RO106-9920 co-treatment. In human tissues, coronary artery medin strongly correlated with plaque burden (R=0.76, p<0.0001) and coronary macrophage content (R=0.72, p<0.0001). Coronary arteries from decedents with myocardial infarction had higher medin than those without (5.53{+/-}2.67% versus 0.02{+/-}0.02%, p=0.0005). ConclusionsMedin induced NF{kappa}B-mediated endothelial cell pro-inflammatory and prothrombotic activation and was strongly associated with coronary plaque burden and inflammation. Medin is a novel candidate mediator linking aging and coronary atherosclerosis.

physiology↗

bioRxiv2025

Can AlphaFold3 predict unseen amyloid polymorphs?

Amyloid fibrils are highly ordered, aggregation-prone protein assemblies implicated in numerous neurodegenerative diseases. Their formation arises from protein misfolding, producing structurally distinct polymorphs that have historically been difficult to predict computationally. While AlphaFold2 excels at predicting monomeric globular proteins, it struggles with protein complexes. Here, we show that AlphaFold3 provides a new opportunity to model amyloid structure and capture polymorph heterogeneity. Using TM-score based clustering of known -synuclein fibril structures, we establish a polymorph nomenclature and demonstrate that AlphaFold3 can reproduce known fibril architectures and predict alternative conformations. These results highlight AlphaFold3s potential for studying amyloid proteins, enabling systematic investigation of amyloid polymorphism.

bioinformatics↗

bioRxiv2025

Medin drives Aβ40 to adopt Aβ42-like fibril polymorphs in vitro

Medin, a vascular amyloid derived from MFG-E8, is the most prevalent form of localized human amyloid and co-localizes with A{beta} in Alzheimers disease and, in particular, cerebral amyloid angiopathy (CAA). While it was shown that medin can promote A{beta} aggregation, it remains unclear whether this amyloid-amyloid interaction affects the structure of the resulting fibrils. Here, we investigate how medin modulates A{beta}40 fibril assembly in vitro using cryo-electron microscopy, aggregation kinetics, and immunogold electron microscopy. We show that medin accelerates A{beta}40 aggregation, co-assembles into hybrid fibrils, and modulates fibril morphology. Cryo-EM analysis reveals two fibril populations: one corresponding to a previously described in vitro A{beta}40 morphology, and a second, previously unobserved polymorph with A{beta}42-like features, including a structured N-terminus and a compact hydrophobic C-terminal core. The presence of a peripheral, unresolved cryo-EM density near the fibril surface suggests that the new polymorph is stabilised through heterotypic interactions, yet the atomic details remain unresolved, likely due to substantial structural heterogeneity. Rather than representing a limitation, this highlights how not all determinants critical for fibril assembly are necessarily ordered or resolvable in the final fibril structure, reflecting the inherent dynamic and heterogeneous nature of amyloid interactions. Our findings provide structural evidence that heterotypic co-aggregation can redirect A{beta}40 into distinct conformational states and suggest that dynamic or transient interactions contribute to fibril polymorphism beyond what can be fully captured in static structural models.

biochemistry↗

bioRxiv2024

Medin Induces Pro-Inflammatory Activation of Human Brain Vascular Smooth Muscle Cells

BackgroundMedin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimers disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. MethodsVSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 {micro}M) without or with small molecule nuclear factor-{kappa}B (NF{kappa}B) inhibitor RO106-9920 (10 {micro}M) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). ResultsMedin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0{+/-}12.8x, 20.2{+/-}4.1x and 8.7{+/-}3.1x, respectively, medin 5 {micro}M versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. ConclusionsMedin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NF{kappa}B. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.

molecular biology↗

bioRxiv2024

Investigating Medin Cleavage Accessibility in MfgE8: Conformational Insights Derived from Molecular Dynamics Simulations and AlphaFold2 Models

Recent studies have indicated that the human amyloidogenic protein medin is associated with a range of vascular diseases, including aortic aneurysms, vascular dementia, and Alzheimers disease. Medin accumulates in the vasculature with age, leading to endothelial dysfunction through oxidative and nitrative stress and inducing pro-inflammatory activation. Medin is a cleavage product from the C2 domain of MfgE8. The exact mechanism of medin production from MfgE8 is unknown, with crystal structures of homologous C2 domains suggesting that the cleavage sites are buried, requiring a conformational transition for medin production. Molecular dynamics simulations can explore a wide range of conformations, from small-scale bond rotations to large-scale changes like protein folding or ligand binding. This study employed a combination of full-atom and coarse-grained molecular dynamics simulations, along with CONCOORD- and AlphaFold2-generated models, to investigate MfgE8 conformations and their implications for medin cleavage site accessibility. The simulations revealed that MfgE8 tends to adopt a compact conformation with the RGD motif, important for cell attachment within the N-terminal domain, and the medin region in the C-terminal domain close in proximity. Formation of this compact structure is facilitated by interdomain electrostatic interactions that promote stability and in turn decrease the solvent-accessible surface area of the medin region and particularly the C-terminal medin cleavage site. This data enhances current knowledge on medin generation to propose that alterations in local environmental conditions, possibly through changes in glycosylation or other post-translational modifications are required to induce MfgE8 to unfold partially or fully: this would result in enhanced accessibility of the cleavage sites and therefore enable medin generation.

bioinformatics↗

bioRxiv2021

Development of amyloid beta gold nanorod aggregates as optoacoustic probes

Propagation of small amyloid beta (A{beta}) aggregates (or seeds) has been suggested as a potential mechanism of Alzheimers disease progression. Monitoring the propagation of A{beta} seeds in an organism would enable testing of this hypothesis and, if confirmed, provide mechanistic insights. This requires a contrast agent for long-term tracking of the seeds. Gold nanorods combine several attractive features for this challenging task, in particular, their strong absorbance in the infrared (enabling optoacoustic imaging) and the availability of several established protocols for surface functionalization. In this work, polymer-coated gold nanorods were conjugated with anti-A{beta} antibodies and specifically attached to pre-formed A{beta} seeds. The resulting complexes were characterized for their optical properties by UV/Vis spectroscopy and multispectral optoacoustic tomography. The complexes retained their biophysical properties, i.e. their ability to seed A{beta} fibril formation. They remained stable in biological media for at least 2 days and showed no toxicity to SH-SY5Y neuroblastoma cells up to 1.5 nM and 6 M of gold nanorods and A{beta} seeds, respectively. Taken together, this study describes the first steps in the development of probes for monitoring the spread of A{beta} seeds in animal models.

biochemistry↗