DTA

Dottorato

CORBANI, EDOARDO

etd-09302025-222853

Identification of Low Molecular Weight Endothelium-Protective Plasma Factors

MED/11

Istituto di Scienze della Vita - PHD IN MEDICINA TRASLAZIONALE

relatore Prof. RECCHIA, FABIO ANASTASIO

• Anti-oxidant metabolites
• Comparative biology
• Endothelial dysfunction
• LDL oxidation

14/07/2026;

parziale

Atherosclerosis is a chronic inflammatory disease characterized by the development of plaques in the arterial intima, primarily driven by endothelial dysfunction. Its pathogenesis comprises inflammation, increased ROS production, upregulation of adhesion molecules (VCAM-1/ICAM-1), and foam cell formation, together leading to progressive plaque formation. Different animal models were used to study this pathology, and investigators found that dogs are very refractory to atherogenesis, even under a high-cholesterol diet. Therefore, given the pivotal role exerted by endothelial damage in atherosclerosis development, we have hypothesized the presence of circulating low molecular weight endothelium-protective factors in dogs, different from the classic protective factors like HDL or other macromolecules known in the literature. Since atherosclerosis is an age-related pathology, we extended our hypothesis to the presence of age-dependent interspecies differences in endothelium protection. EDTA-plasma/serum pools from dogs, pigs, and young (<30 years) or old (>65 years) humans, all males, were subjected to 10 or 3 kDa ultrafiltration and subsequent size-exclusion chromatography. To investigate endothelial protective activity, we tested the capacity of ultrafiltrates to slow down copper sulphate- or ammonium-persulfate (APS)-mediated low-density lipoprotein (LDL) oxidation and counteract VCAM-1 upregulation induced in cultured human aortic endothelial cells (HAoEC) by TNFα (1-10 ng/mL). Dog EDTA plasma 10 kDa ultrafiltrates exerted the most endothelium-protective activity among the experimental groups, being able to significantly reduce TNF-α-induced VCAM-1 upregulation in HAoEC and exert the most antioxidant capacity towards LDL. Size exclusion chromatography fractionation allowed us to associate this protective activity with the fraction peak 2C (the latter portion of the second peak visualized in the 280 nm chromatogram), which maintained its activity even after further ultrafiltration (3 kDa) and heating. Confounding data emerged regarding the role of EDTA (present in EDTA-plasma ultrafiltrates) in endothelial protection and antioxidant activity, as LC-MS/MS analysis revealed high concentrations of this anticoagulant in the most protective fraction 2C, consistent with its potent metal-chelating properties, including copper. Subsequent experiments demonstrated the persistence of these protective antioxidant effects when using APS, a molecule not directly chelated by EDTA, prompting us to use serum as the test material for ultrafiltrates to fully eliminate confounding effects of EDTA. Dog serum ultrafiltrates proved the most protective among the species tested, with fraction 2C retaining the highest endothelial protective activity. Metabolomic analyses identified elevated concentrations of 3-methylhistidine in fraction 2C; this metabolite exhibited strong antioxidant activity against LDL at supra-physiological concentrations, although it did not significantly reduce TNF-α-induced upregulation of VCAM-1 in HAoEC. When serum ultrafiltrates were tested individually, intraspecies variability was observed, but not correlated with any of the metabolite concentrations. Subsequent experiments testing the LDL antioxidant power of serum ultrafiltrates of patients with coronary heart disease (CAD) versus healthy subjects (all males) did not show significant differences between the two groups, despite some differences in individual cases. Collectively, these results partially support the working hypothesis, particularly relative to protective factors counteracting LDL oxidation. Inter- and intraspecies differences exist in the endothelial protective capacity of circulating factors. Several candidate molecules emerged; however, none fully accounted for the total protective effect observed on both endothelium and LDL, hence further studies are warranted.

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